Barnsley are seven points adrift of the play-off places after a 3-1 loss at Colchester United on Monday.
George Waring claimed the opening goal just before half-time but the relegation-threatened hosts netted three times in the final half an hour.
The Reds are now down to 11th and may need to win all five of their remaining games, including a home match with sixth-placed Chesterfield on Saturday, in order to sneak in the top six.
Head coach Lee Johnson made four changes to the side that lost 2-0 at home to Sheffield United 48 hours earlier. Goalkeeper Adam Davies returned from injury with Joe Wildsmith dropping out of the squad.
Jabo Ibehre was not allowed to play against his parent club so Waring deputised for him up front while Milan Lalkovic returned from suspension to replace Cameron Stewart and James Bailey came in for Sam Winnall.
The Reds played a 4-1-4-1 formation with Bailey in front of the back four while Lalkovic and Josh Scowen were on the wings.
While Barnsley dominated possession in the first half and played some good football, they struggled to create any real chances until they netted in the 40th minute. Conor Hourihane swung in a fine free-kick from the right wing and, after Lewin Nyatanga flicked the ball on, Waring appeared to get the final touch for his sixth of the season.
Colchester started the second half a lot brighter with Chris Porter testing Davies from a tight angle then, seconds later, Alex Wynter headed straight at the Reds goalkeeper with the goal gaping when unmarked inside the six yard box.
Ben Pearson forced home goalkeeper Sam Walker into a good save with a curling shot from the edge of the box but Barnsley then fell apart.
Colchester levelled on 62 minutes when Gavin Massey was left free to tap in a Jacob Murphy right-wing cross inside the box at the back post.
Massey returned the favour with ten minutes to go as he crossed from the left for Murphy to head in.
Substitute Sam Winnall thought he had levelled when he rounded Walker and netted on 85 minutes but the linesman controversially ruled it out for offside.
George Moncur made sure of the home win late on when he fired in from the edge of the box after Massey's fine run and pull-back.
