ATP world number four Tsitsipas is one of the headline names in action at the event, which is acting as a warm-up tournament for the Australian Open.
After going a set down to his Bulgarian opponent in his first game of the inaugural tournament, Tsitsipas hit back with a strong second set before holding his nerve in a tense tie-break in the decider, securing a 4-6 6-2 7-6 (7-4) win.
Tsitsipas's team-mate Despina Papamichail continued Greece's strong start, also coming from a set down to beat Isabella Shinikova 3-6 6-4 6-1.
Great Britain opened with a pair of victories, Cameron Norrie and Katie Swan earning straight sets triumphs over their Australian counterparts in Sydney.
Norrie, who was initially set to play Nick Kyrgios prior to the Wimbledon finalist's withdrawal due to an ankle injury, eased to a 6-3 6-3 victory over Alex De Minaur, while Swan defeated Zoe Hives.
The United States are also taking a 100 per cent record into day two of the tournament, after Taylor Fritz and Madison Keys overcame Jiri Lehecka and Marie Bouzkova respectively, while France pair Arthur Rinderknech and Alize Cornet got off to winning starts against Argentina's Francisco Cerundolo and Maria Lourdes Carle.
In Brisbane, Swiss hope Belinda Bencic secured a 7-6 (7-0) triumph over Yulia Putintseva of Kazakhstan and Beatriz Haddad Maia, the Brazilian world number 15, dispatched Italy's Martina Trevisan 6-2 6-0.
Former world number one and third seed Williams prevailed in a tricky test against Margarita Gasparyan in straight sets in New York on Thursday.
Countrywoman and second seed Kenin contained Canadian teenager Fernandez behind closed doors at Flushing Meadows.
It was not good news for 10th seed Muguruza, who was sent packing by unranked Bulgarian Tsvetana Pironkova.
SERENA WINS THROUGH
Williams continued her quest for a record-equalling 24th grand slam after defeating Russian Gasparyan 6-2 6-4 to set up a showdown with 2017 US Open winner Sloane Stephens.
The 38-year-old Williams hit 27 winners and just 16 unforced errors, an improvement on her numbers in the opening round, under the Arthur Ashe Stadium lights.
Williams, who has not won a slam since the 2017 Australian Open, will next meet Stephens after the American 26th seed eased past Olga Govortsova 6-2 6-2.
KENIN SEES OFF FERNANDEZ
Kenin advanced to the third round of her home slam for the fourth year in a row thanks to a 6-4 6-3 victory over Fernandez.
World number four Kenin did not face a break point in a contest lasting one hour, 21 minutes.
After firing 19 winners and three aces, Kenin will play Ons Jabeur – who took down Kaia Kanepi 7-6 (10-8) 6-0.
MUGURUZA STUNNED
Muguruza's US Open campaign came to an end in the second round, sensationally upstaged 7-5 6-3 by Pironkova, who is playing her first tournament since Wimbledon in 2017.
Australian Open runner-up and two-time slam champion Muguruza was serving for the first set when the Spaniard started to unravel.
Pironkova, who gave birth to a son during her time away from the WTA Tour, took control at the end of a tight first set and never looked back.
"I'm not going to hide, I didn't expect my first tournament to be so good and to play so well. It's welcome! That's why I came back, for these matches and these stages," Pironkova said after winning 93 per cent of her first serves.
AZARENKA ROLLS ON
Former world number one and two-time Australian Open champion Victoria Azarenka topped fifth seed Aryna Sabalenka 6-1 6-3.
A US Open finalist in 2012 and 2013, Azarenka is in fine form after winning the Western & Southern Open last week.
And Azarenka is now just one victory away from her first second-week appearance at Flushing Meadows in five years.
Meanwhile, seventh seed Madison Keys also progressed to the third round.
Gauff reached the second week at Flushing Meadows in the women's singles for the first time after cruising to a 6-2-6-3 win over Madison Keys on Friday.
The number 12 seed saw off her fellow American in composed fashion to book her place in the last 16, exceeding her previous best result at the US Open in 2019.
Gauff was largely in control against the number 20 seed Keys to deliver near-flawless performance, chasing a second grand slam final of the year after finishing as runner-up at the French Open to Iga Swiatek.
She will face China's Zhang Shuai in the next round, but when asked if she will watch Williams' match against Ajla Tomljanovic later in the day during her on-court interview, Gauff said she would, adding: "It's been amazing watching Serena's matches so far. I don't know when I'd be due to face her but that's the goal.
"It's been a lifelong dream of mine to do that. Five years ago yesterday I was watching Venus and Serena play here. It's crazy to be on this court now.
"I tried to flex to my friends that I had courtside seats, now I'm on the court!"
Should both remain in the tournament, Gauff and Williams would face each other in the semi-finals.
Gauff also paid tribute to Keys, and to the crowd at Arthur Ashe Stadium, saying: "[The win] means a lot to me... I missed my alarm and was so late. I was here until 9pm last night and lost a doubles match [with Jessica Pegula].
"Kudos to my team, they got me up for it.
"I wasn't sure how the crowd was going to go. We all love Madison... I'm good friends with her off the court, but having support here meant a lot."
Vondrousova's topsy-turvy match against Katie Volynets started on Wednesday before concluding on Thursday, with the number five seed coming out on top 0-6, 6-1, 6-4.
The American started strongly, storming through the first set, but Vondrousova recovered well to take a commanding 4-1 lead before rain washed out the rest of the game.
Vondrousova picked up where she left off upon resumption on Thursday and won one game before another delay due to rain, but eventually saw out the win.
Keys, meanwhile, won in straight sets against Mayar Sherif, holding out for a 6-0 7-6 (9-7) victory.
The American cruised through the first game, enjoying three breaks, but a stern fightback from Sherif almost caused some problems.
Keys held her nerve though, avoiding a decider by saving three set points to prevail on Court Suzanne Lenglen.
Vondrousova will face Chloe Paquet in the next round, while Keys will play either Sara Errani or Emma Navaro.
Data Debrief: Vondrousova marches forward
The Roland Garros is the tournament where Vondrousova has won the joint-most career matches (13, equalling Indian Wells).
She is also unbeaten against opponents ranked above 100 in the WTA since the start of 2023, going 10-0 against such competitors.
Williams needed just 44 minutes to crush 18-year-old Amanda Anisimova 6-1 6-1 in their last four clash at the WTA International tournament in New Zealand.
The 23-time grand slam champion mixed 17 winners with six unforced errors and lost just nine points on serve. Williams served five aces and converted all five of her break points in a dominant display in the lead up to the year's first grand slam in Melbourne.
The American moved into her 98th WTA Tour final and will be aiming for a 73rd title but first since the 2017 Australian Open when she faces Jessica Pegula.
Pegula upset fifth seed Caroline Wozniacki 3-6 6-4 6-0 in their semi-final.
A two-time runner-up, Wozniacki – who will retire after the 2020 Australian Open – won just 10 points in the third set.
Wozniacki will contest the doubles final with Williams against Taylor Townsend and Asia Muhammad.
In Brisbane, Pliskova lost a first set on a tie-break in which she squandered two set points and saved four, and was staring down the barrel after Osaka broke for a 6-5 lead in the second.
However, Osaka's first serve on match point was overly tentative and a series of errors handed Pliskova the break back. The second seed took full advantage, winning the subsequent tie-break and taking the decider in comfortable fashion to defeat the reigning Australian Open champion 6-7 (10-12) 7-6 (7-3) 6-2.
Earlier, Madison Keys rallied past Petra Kvitova to clinch a 3-6 6-2 6-3 victory, meaning there will be no all-Czech final in Queensland.
At the Shenzhen Open, Russian Ekaterina Alexandrova clinched her maiden WTA Tour title with a 6-2 6-4 win over Elena Rybakina in the final.
French Open finalist Paolini progressed to the last eight with a 6-3 6-7 (6-7) 5-5 victory, though the Italian was on the brink of an exit before that.
The seventh seed trailed 5-2 in the deciding set before dragging back into the contest by winning two games, with Keys then calling for the physio and a medical time-out due to a thigh issue.
American Keys was visibly distraught after leaving the court for treatment, returning to concede another game and struggling to serve before retiring with the game finely poised.
Paolini will now face either Emma Navarro or Coco Gauff in the quarter-finals at SW19.
Data Debrief: Paolini's Italian pride
Paolini became the first Italian in the Open Era to reach the women's singles quarter-final at Wimbledon and Roland-Garros in the same season, having fallen in the French Open final to Iga Swiatek.
Having never won a match at Wimbledon before this year, Paolini has now won four straight, though this triumph came in unwanted circumstances.
Keys is only the second player in the Open Era to retire in the third set of a women's singles round-of-16 clash at a major, after Julie Heldman progressed past Billie Jean King via retirement at the US Open in 1973.
