The Australian duo came from a set down to win on Court Philippe Chatrier in just over two hours, emerging 6-7 (6-8) 7-6 (7-1) 10-8 victors at Roland-Garros.
Krajicek and Ram would start well, winning a break point in the sixth game, only for their Australian opponents to respond instantly to level the encounter at 4-4.
With both pairs holding their serve for the remainder, a tie-break would decide who would take the first set, with the Americans able to obtain the early advantage.
Krajicek and Ram found another early break in the second, but again saw their lead vanish as Ebden and Peers rallied to win three consecutive games.
The Australian's took the game to a second tie-break, producing some stunning tennis along the way as the encounter went the distance in the French capital.
Ebden and Peers would carry their momentum into the decider, and despite missing four gold medal points, were able to hold on to secure the triumph.
Data Debrief: Australia strike gold at Roland-Garros
Ebden and Peers showed grit and determination worthy of winning a gold medal, coming from a set down to scoop the top prize.
The Australian's impressed on serve, scoring nine aces compared to Krajicek and Ram's six, winning 73% of their first-serve points.
The world number one saw her hopes of winning a gold medal for Australia at Tokyo 2020 brought to an end on Friday.
Barty and partner John Peers won the first set against Russian Olympic Committee duo Andrey Rublev and Anastasia Pavlyuchenkova.
But Rublev and Pavlyuchenkova turned it around to win an epic clash 5-7 6-4 13-11 and reach the final.
With Barty already eliminated in the singles and women's doubles competitions, the best she can now hope for is a bronze medal alongside Peers.
They will meet Serbia's Novak Djokovic and Nina Stojanovic for the last spot on the podium.
"It is a tough one to swallow," said Barty. "I think those tie-breaks at times, they go either way and we just didn't quite have the run of the grain.
"We put ourselves in a position to win the match, we just weren't able to close it out. It was a good level, no doubt disappointing."
Another chance at a medal means Wimbledon champion Barty will try to get over her loss swiftly.
She added: "But we get to fight for a medal - this one hurts but we still have an opportunity to play another match and try to win a medal for Australia.
"We pick ourselves up pretty quickly, move on and know that we will give it our all and keep fighting right until the end.
"It's been quite a while since we've had an Australian medallist here [in tennis] at the Olympics so we're looking forward to that challenge."
In the men's doubles, Nikola Mektic and Mate Pavic are Olympic champions after they beat fellow Croatia representatives Marin Cilic and Ivan Dodig in the final.
