A goal in either half from Jamille Matt and Josh March earned Forest Green Rovers all three points against the U's at the Fully Charged New Lawn in the latest league match.
Jamille Matt put the home side ahead in a largely uneventful first half. But Mullins' side fought back in the second half, going close on a few occasions without being able to break through a tough Rovers' defence.
Josh March put the icing on the cake in second-half stoppage time when he slotted the ball beyond Jake Turner to ensure the home side would keep all three points.
Hayden Mullins named a line up with one change from the U's last game, last Tuesday's Papa John's Trophy game against Sutton United.
Mullins handed Corie Andrews his first start since signing on loan for the rest of the 2021/22 season, with Armando Dobra named on the bench. Cam Coxe was also named in the starting eleven, for the first time since joining the club on a permanent basis, with Tom Dallison listed as a substitute.
Noah Chilvers was also named in the starting line up for the U's, with Freddie Sears and Armando Dobra both missing from the squad with illness.
The U's were pinned almost in their own half for the opening ten minutes of the game, with the league leaders looking to show why they are the favourites for promotion. The back three of Eastman, Chambers and Smith did well to contain their attacking counterparts, protecting Jake Turner well.
And Hayden Mullins' side looked to take the advantage up the other end, with Alan Judge going close. Noah Chilvers picked up the loose ball from the Rovers' defence, backheeling it to Judge who looked to bend on into the far corner, but Luke McGee was able to get across.
But with quarter of an hour played, the home side got their noses in front, and it was their top scorer, Jamille Matt, adding to his tally for the season. Udoka Godwin-Malife cut the ball back to the Rovers captain, who flicked an effort beyond Turner and into the back of the net.
For the remaining half hour of play, there was very little goal-mouth action and not another shot on target. The closest anyone would come to getting another goal came from an unlikely effort by Cole Skuse.
The U's midfielder had latched on to Noah Chilvers' cut back cross, cleverly left by Junior Tchamadeu, seeing his low effort deflected wide and out for a corner.
And althogh the home side had the greater share of possession in the first period, and looked comfortable on the ball, it was all still to play for in the second half of the match, with Rovers' slender one-goal advantage separating the two teams.
It was a lead that looked under threat at the start of the second half, with the U's pressing hard to get the equaliser in the game, forcing Luke McGee into a couple of good saves in the opening ten minutes.
The first came after a neat move by Junior Tchamadeu, Brendan Wiredu and Corie Andrews. The wing-back and central midfielder combined to play the ball through to the U's on-loan striker, with Andrews' effort fired straight at the home 'keeper.
McGee was called into action again from the resulting corner, when Junior Tchamdeu's recycled cross was only half cleared out to Noah Chilvers. Getting a clean strike on the ball from just outside the penalty area, Chilvers saw his driven effort palmed away by McGee.
Cole Skuse again went close to levelling things up with just over an hour gone. The experienced midfielder nipped in to pinch the ball from Hendry, running at goal and shooting low to force the home 'keeper into another save, although this one was al ittle more routine.
It looked as though it might be game over with just over fifteen minutes to play for the U's when Forest Green and Jamille Matt thought they had their second. The home captain carefully controlled the ball from Wilson's delivery, slotting past Turner, but for the linesman's flag to be raised for offside.
The U's went incredibly close to taking what would arguably have been a deserved point, when Luke Hannant was played through by Cameron Coxe. The substitute was able to beat the 'keeper with his low effort, but not the retreating Jordan Moore-Taylor who managed to prevent an equaliser on the goal line.
Forest Green Rovers added a second in stoppage time, with substitute Josh March slotting the ball beyond Jake Turner with a low effort, ensuring the points would be staying in Gloucestershire.
The defeat means the U's stay twenty-second in the table with twenty-two points from twenty-two games.
Up next is a home match against Barrow on Saturday afternoon, with kick off at 3pm at the JobServe Community Stadium.
Forest Green Rovers starting XI: McGee, Wilson, Bernard, Stevenson, Stevens (March 87'), Aitchinson (Young 79'), Cadden, Matt, Moore-Taylor, Hendry (Sweeney 79'), Godwin-Malife.
Unused subs: Thomas, Cargill, Edwards, Diallo.
Colchester United starting XI: Turner, Chambers, Smith, Wiredu, Skuse, Judge (Tovide 80'), Chilvers (Hannant 68'), Andrews, Eastman, Tchamadeu (Daniels 68'), Coxe.
Unused subs: Gerken, Dallison, Kennedy, Cooper.