Assistant coach Ferran Sibila revealed the Reds had only 12 fit players in training today and were without head coach Poya Asbaghi, who had a sore throat but has twice tested negative for Covid-19 since he felt it during Saturday's 2-1 defeat at Birmingham City.
Barnsley therefore asked for their rearranged match against Nottingham Forest to be postponed a second time, but the Football League (EFL) refused.
The playing absentees included an unspecified number of players who were unable to train because of knocks but have not yet been ruled out of the trip to the City Ground. Sibila was unable to say if there might be a second request to postpone if some of those are definitively ruled out.
Football League rules say games can be postponed if teams have fewer than 13 outfield players and/or no goalkeeper available. The rules are intended to protect teams against Covid-19 outbreaks but the way some teams have used them to cover other absentees has been criticised.
"Today (Monday) in training we had less than 13 players from the first team available for the match," said Sibila. "We tried to postpone the game but we were not allowed.
"I don't know exactly the communication between the club and the EFL, the only part I know is we will play the game.
"From the last game we know we have Liam Kitching out because he has had a concussion, then (Callum) Styles. These are the players from the last game and after you play a game that is tough physically, players have different knocks so today they were not able to have proper training. Let's see how they wake up tomorrow.
"Styles is a question (mark) but it gives opportunities to other players. It's not a situation we want but the new players come in with lots of energy and every player who has come into training has done so with a lot of ambition to prepare 100 per cent for the game, so that is what we will do."
Josh Benson, Callum Brittain and Clarke Odour are all still missing but Sibila said Aaron leya Iseka was "100 per cent to play".
Sibila was not prepared to say how many of the absentees had Covid-19, how many were isolating as close contacts, and how many injured.
Adding to Barnsley's frustration is the fact this game has already been postponed once because of Covid cases in the Forest squad.
"We remember the 3rd of January we were due to play against Nottingham," said Sibila. "We wanted to play the game but Nottingham had the chance to postpone because of Covid cases even though they have more than 30 players in their squad but today we are not able to postpone our game with less than 13 players in our squad for training today."
January's games against Stoke City (itself rearranged from Boxing Day) and Blackpool were also postponed because of a lack of numbers - the first on Barnsley's part, the second Blackpool's. It has been an issue throughout English professional football since mid-December.