Goals from Liam Kelly and Danilo Orsi saw the U's beaten 2-1 at home by Crawley Town.
The U’s were down a goal at the break, after Liam Kelly slotted home from inside the penalty area just before half time.
Twenty minutes into the second period, the visitors doubled their lead after Danilo Orsi converted from the penalty spot.
Colchester fall to twenty-second in the standings, five points above the drop zone, facing a trip to promotion-chasing Wrexham next weekend.
Matty Etherington named a side with three changes from Tuesday’s defeat to West Ham United, with Owen Goodman, Cam McGeehan and Joe Taylor all returning to the starting eleven; out of the team were Tom Smith, Gene Kennedy and Bradley Ihionvien.
Matt Jay was given his first start in the league since April, with Owen Goodman back in the team after recovering from an ankle injury.
Joe Taylor and Samson Tovide lead the line-up with Matt Jay sitting just behind. Arthur Read and Cameron McGeehan started just behind, flanked by the pair of Will Greenidge and Jayden Fevrier. Egbo, Mitchell and Kelleher made up a three-man defence in front of Owen Goodman.
Samson Tovide was given a good chance within the opening five minutes, with Joe Taylor delivering a dangerous ball to attack in the centre of the penalty area; he couldn’t connect as the ball slid out for a Crawley throw in.
The U’s soon went close from a free kick, with Arthur Read finding the roof of the net, before Samson had another opportunity to open the scoring. This time, Will Greenidge was the man delivering, but Tovide got too contact with the ball flying out for a goal kick.
Crawley soon had a chance of their own with twenty minutes on the clock. Liam Kelly should have done better from Gordon’s pull back, but the Irish midfielder mis-cued his effort well wide of the mark.
Kelly had another shot ten minutes later that he should have done better with. The U’s failed to deal with a looping cross, with Orsi able to divert the ball back to a shooting position, but Kelly shot high and wide.
Cameron McGeehan picked up the first card of the afternoon for a foul on Gladwin, despite slipping on the surface when attempting to win the ball back.
The visitors took the lead with five minutes of the half to go when Kelly got an effort on target, squeezing the ball in at the near post despite Goodman’ touch on the effort.
Jayden Fevrier was booked just before half time after an innocuous challenge on Gordon, as the U’s trailed 1-0 at the break.
The U’s opted not to make any changes at the half, but were soon forced into one when John Akinde return to action in the fifty-fourth minute. Samson Tovide collided with Dion Conroy, with the U’s man needing help to leave the pitch after picking up an injury.
But the challenge became tougher for Matty Etherington’s side ten minutes later when Crawley added to their tally. Danilo Orsi had the ball in the net before the referee pulled play back to award the visitors a penalty for Arthur Read’s challenge in the area. Orsi stepped up to convert from twelve yards.
The visitors made a swift double change with Gordon and Tsaroulla both replaced by Darcy and Forster for the final twenty-five minutes.
Laurence Maguire went into the book for the visitors after a late challenge on Will Greenidge, but the visitors were seemingly in control.
Two more changes followed for the visitors with goalscorer Kelly and Campbell replaced by Lolos and Adeyemo for the last twenty minutes, while the U’s also made changes.
Will Greenidge and Matt Jay left the pitch with Noah Chilvers and Chay Cooper brought on to influence the last fifteen.
The U’s threw everything to try and get back in the game, with Joe Taylor netting a goal in the sixth minute of eleven of injury time - racing through on goal he poked the ball over the onrushing 'keeper.
But it wasn't enough as the U's fell to a 2-1 defeat.
Colchester fall to twenty-second in the standings; up next for Matty Etherington’s side is a trip to promotion-chasing Wrexham next weekend.
Colchester United starting XI: Goodman, Greenidge (Chilvers 76’), Kelleher, Jay (Cooper 76’), Tovide (Akinde 54’), McGeehan, Mitchell, Read, Egbo, Taylor, Fevrier.
Subs: Smith, Bandeira, Chilvers, Hopper, Akinde, Cooper, Terry.
Crawley Town starting XI: Ashby-Hammond, Gordon (Darcy 66’), Conroy, Kelly (Lolos 72’), Maguire, Gladwin, Orsi, Tsaroulla (Forster 66’ (Simon-Swyer 88’)), Williams, Campbell (Adeyemo 72’), Wright.
Subs: Addai, Lolos, Darcy, Forster, Adeyemo, Johnson, Simon-Swyer.
Attendance: 3,187 (257)