Conor Coady and Marcus Rashford have been ruled out of England's Nations League matches against Belgium and Iceland.
The Three Lions kicked off November's triple-header with a 3-0 friendly win against the Republic of Ireland on Thursday thanks to goals from Harry Maguire, Jadon Sancho and Dominic Calvert-Lewin.
It was as smooth an evening as England could have hoped for, but Southgate will be without suspended Maguire in Belgium on Sunday and impressive Reece James for both remaining Nations League games.
Wolves defender Coady and Manchester United forward Rashford have also been ruled out of the upcoming matches, whittling the Three Lions' options down even further.
In a statement, the Football Association said: "Conor Coady and Marcus Rashford will miss England's remaining November internationals.
"Coady has been forced to self-isolate having discovered he had inadvertently come into contact with somebody prior to camp who has tested positive for Covid-19 via an NHS Test and Trace alert.
"The Wolverhampton Wanderers defender had returned two negative tests as part of The FA and UEFA's protocol but, in line with UK Government guidelines, has to adhere to a period of self-isolation.
"Meanwhile, Rashford's arrival into camp had initially been delayed to allow for further assessment on an injury sustained last weekend but the Manchester United forward will now not meet up."
Wolves skipper Coady says he is "absolutely gutted" to have to pull out of the England squad.
"I contracted and recovered from the virus myself back in March, and have tested negative twice in camp, but of course it is important that I abide by the rules set by the government and observe a period of self-isolation," the defender said.
"It's so frustrating to have to miss games because of this, but these are the rules that are set out and the most important thing is to make sure my team-mates are not put at risk.
"My next priority will be to keep my fitness and hope I can be back and available for selection for the next Wolves fixture.
"For now, I will be supporting the lads from afar and I want to wish them all the very best of luck for the next two games."
That frustration was shared by Southgate, but the England boss believes he has enough numbers to cope.
"To lose both is disappointing," he said. "We had some doubts about Marcus following the weekend, we hoped he might progress but it hasn't happened.
"Conor's is just the bizarre world we are living in. It is one of those weird ones where he has got to isolate when he is more than likely not a risk.
"It is a great disappointment for him, he was bitterly disappointed because he has had a big impact on the group."
