Alan Judge's superb free kick in second half stoppage time proved to be just a consolation for the U's as they were defeated 2-1 at Mansfield Town.
The U's were a goal down at the break with Rhys Oates' run and strike giving Nigel Clough's side the opener in the first twenty minutes of the game.
The advantage was doubled in second half stoppage time with Jordan Bowery converting from the penalty spot, before Alan Judge netted with a free kick from a tight angle. But the U's were unable to find another as Wayne Brown's side fell to a first defeat in five games.
Wayne Brown named a side with three changes to the eleven that started against Leyton Orient on Saturday afternoon.
Coming into the lineup were Luke Chambers, Alan Judge and Gene Kennedy in place of Cole Skuse, Corie Andrews and Tom Eastman.
The opening quarter of an hour was a fairly even contest between the two sides, although the Stags were finding joy down the wings with the U's struggling to stop the crosses.
Wayne Brown's side looked to hit their hosts on the counter attack, and were effective in getting the ball forward with Hannant and Wright working the channels with Judge.
And the counter attacking football earned the U's a free kick in a good area, but it was a quick break from Mansfield Town that saw them grab the opening goal of the game. Alan Judge's delivery was cleared out to Rhys Oates on the halfway line, with the forward driving forward, and shooting well beyond Hornby into the corner of the goal.
The U's had a good couple of half-chances within the next ten minutes, but were unable to test Bishop in the home goal with any shots on goal. Alan Judge picked out an unmarked Tommy Smith from a corner kick, with the captain getting his header back across wrong, looping it over the bar when he should have done better.
And Junior Tchamadeu kept the pressure on when bursting forward from full back. Luke Hannant spotted the run of the eighteen-year-old, but so had Stephen McLaughlin, who was able to track his man and clear the ball for a corner before Tchamadeu was able to pull the trigger.
The Stags had a golden chance to double their advantage ten minutes before the break, after the referee had adjudged Tommy Smith to have passed the ball back to Sam Hornby, who collected. It was a harsh decision on the U's defence, but it wouldn't matter in the end with former Colchester United player John-Joe O'Toole smashing the ball over the bar from the indirect free kick.
Wayne Brown opted for a change of personnel at half time, with Tom Eastman replacing Luke Hannant. The substitution brought a change of shape, with Eastman slotting in at right centre-half, with Tchamadeu and Kenlock playing at wing-back for the second period.
The second half brought a much tighter affair, with the U's change of shape. Playing a three-man defence helped keep things tight at the back with the Stags only registering one shot on target in the whole of the second period.
There were a couple of dangeous moments at the back for either side, with the U's looking to get back on level terms early on in the second half. Elliott Hewitt was pressured by Tyreik Wright, playing a short back pass to Bishop, with Sears able to get a touch. But the U's top scorer was unable to control it as he shot into the side netting.
At the other end of the pitch, Tom Eastman had to be very careful with Lucas Akins lurking at the back post, and the U's defender got a little bit of luck with his second touch taking the ball away from the Stags' forward before clearing.
Wayne Brown's side really put the pressure on in the final ten minutes of the game, with Sears having a deflected cross collected by Bishop. And Tchamadeu crossed from the other flank, with the home 'keeper collecting under pressure from the onrushing Sears a couple of minutes later.
But as the game entered seven minutes of stoppage time, the home side won a penalty with Tom Eastman fouling Ollie Clarke as the Stags broke quickly. Jordan Bowery sent Sam Hornby the wrong way to make it a two-goal advantage.
Alan Judge did get a goal back for the U's, with a sublime free kick after Corie Andrews had been fouled in a wide position, but it proved to be too little, too late despite a couple of blocked shots in the dying second of the game.
Mansfield Town hand a first league defeat to Wayne Brown, with Nigel Clough's side making it ten games unbeaten in the league.
The U's remain twentieth in the table, with thirty points from twenty-nine games. Up next for Colchester United is a home tie against twenty-second-placed Carlisle United who are three points behind Wayne Brown's side.
Mansfield Town starting XI: Bishop, McLaughlin (Wallace 77'), Hewitt, Murphy (Clarke 73'), Maris (Bowery 82'), Hawkins, Quinn, Oates (Rawson 87'), Stirk, Akins, O'Toole.
Unused subs: Stech, Johnson, Longstaff.
Colchester United starting XI: Hornby, Chambers, Smith, Wireud, Hannant (Eastman 46'), Judge, Sears, Kenlock, Kennedy (Andrews 82'), Tchamadeu, Wright (Edwards 65').
Unused subs: Collins, Welch-Hayes, Coxe, Cornish.