Holgate, who has been a mainstay alongside Michael Keane at the heart of the Toffees defence, picked up a knock to his shin during Monday's defeat to Tottenham Hotspur, which forced him off in the first-half.
Ancelotti confirmed that the 23-year-old will be out for their next game, but anticipates his return for Sunday's trip to Wolves, who are also chasing European football next season.
Holgate set to miss Saints match, but Walcott available
Everton have been given a boost ahead of the Southampton game with Theo Walcott, who hasn't featured for Ancelotti's side since the return of the Premier League, available after returning to full training.
Alex Iwobi is also fit for Thursday's match, but Djibril Sidibe may not be quite ready to play his first full match just yet. Midfielder Fabian Delph remains on the sideline as he is still recovering from a muscle injury.
Ancelotti, speaking about Sidibe in his pre-match press conference, said: "He is not 100 per cent because he didn't train properly in the last 15 days. I don't know if he can do 90 minutes at the top of his intensity."