England duo Mason Mount and Ben Chilwell will miss the match with the Czech Republic and must self-isolate up to and including June 28.
It was confirmed on Monday that the Chelsea players had begun isolating after being deemed close contacts of Billy Gilmour, who tested positive for coronavirus after Scotland's 0-0 draw with England at Wembley last Friday.
The Football Association said the precautions had been taken after consultation with Public Health England, despite both players returning negative PCR and lateral flow test results.
On Tuesday, the FA said Chilwell and Mount will train individually in private areas of England's St. George's Park base, where the rest of the squad will return after their final Group D match with the Czech Republic.
It means that, should England win and top the group, Mount and Chilwell will miss the last-16 match, which takes place on June 29.
If Gareth Southgate's side fail to take three points, they will finish second in the group behind the Czech Republic, meaning their first knockout match will be on June 28.
England said via Twitter: "We can confirm that Ben Chilwell and Mason Mount must isolate up to and including next Monday [28 June]. This decision has been taken in consultation with Public Health England.
"The pair were confirmed overnight as close contacts of Scotland’s Billy Gilmour after his positive test following last Friday's match.
"Chilwell and Mount will isolate and train individually in private areas at England’s training base St. George's Park, with the rest of the squad returning there after tonight's fixture against Czech Republic at Wembley.
"We will continue to follow all COVID-19 protocols and the UEFA testing regime, while remaining in close contact with PHE.
"The entire England squad and staff had lateral flow tests on Monday and all were again negative, as was the case with Sunday's UEFA pre-match PCR tests.
"Further tests will be carried out as and when appropriate."