Elliott List's first half penalty was enough to separate the two teams as Stevenage ran out 1-0 winners at the Lamex Stadium.
Colchester United started the game brightly, but were unable to convert their chances, with the hosts taking full advantage from their first half injury time penalty.
Frank Nouble had the ball in the back of the net for Hayden Mullins' side shortly after the break, but the linesman had his flag up for offside, with real goalscoring chances a premium for Colchester United in the second period.
Hayden's side pressed until the final whistle, but couldn't find the opening and fell to a 1-0 defeat.
The U's lined up with four changes from their last match, ten days ago against Ipswich Town.
Shamal George, Cole Skuse, Armando Dobra and Sylvester Jasper returned to the starting eleven, with the latter two back in the squad after international duty. Jake Turner and Luke Hannant dropped to the bench, with Gene Kennedy and Brendan Wiredu both missing through injury.
Colchester United started the game brightly, with Noah Chilvers and Cole Skuse pulling the strings in the centre of midfield.
With the attacking runs from Sears, Dobra, Jasper and Nouble ahead of them, the midfielders were able to pick out some clever passes from the middle of the park and create shooting opportunities for the attackers.
But it was the hosts who had forced the first save of the game out of one of the 'keepers. Jake Taylor was the man on target, after a clever run from just outside the area, but Shamal George was able to rush out and claim the ball.
Then between the fifteen minute mark to half time, it was Hayden Mullins' side who were looking the most likely to find the back of the net, despite ultimately trailing at half time.
Noah Chilvers picked out the run of Frank Nouble with a driven ball between the full back and centre half of the home side, with Nouble able to deliver a cross to the front post. Cuthbert was able to get a touch to Sears' toe-poke, pushing the ball wide from the six-yard line.
Sylvester Jasper looked like a livewire on the right flank, attacking Ben Coker and Max Melbourne with dangerous runs towards goal, twice going close before the half hour mark.
His first effort forced a routine save by Adam Smith, with the ball at a kind height, but Jasper's second brought out a brilliant save from the home 'keeper five minutes later. A similar run saw the Fulham loanee's effort curl towards the far right top corner, but Smith clawed the ball away for a corner.
With fifteen minutes to go in the first period, the chances started to become less frequent for the U's, as the home side managed to settle into shape.
Stevenage were able to pick some better balls out, testing George in goal with crosses from both wings, before they made the breakthrough in the game just before the teams headed to the changing rooms.
Elliott List won and took the penalty that would put the home side ahead at half time, after Shamal George was booked for bringing the pacey forward down on the edge of the area.
After half time, the U's had to make a change, with Shamal George going off injured after sustaining a knock in the penalty incident.
It took Hayden Mullins' side three minutes to find the back of the net, with Frank Nouble on target for the visiting side. His accurate header seemed to have his side back on level terms, but the linesman's flag was up for offside and the U's were still trailing.
Colchester United were doing their best to get back on level terms, but struggling to really test Smith in the home goal, as they had done in the first.
There were crosses aplenty for Sears and Nouble to attack from Tchamadeu and Daniels, but the strong defensive partnership of Cuthbert and Melbourne were able to hold as time ran out for Colchester United.
Defeat for the U's ends their three game unbeaten run, as Hayden Mullins' side drop to twentieth in the Sky Bet League Two table, level with the hosts on seventeen points.
Up next for the U's, Exeter City make the journey to the JobServe Community Stadium, fresh off the back of a 2-1 win against Carlisle United at home on Saturday afternoon.
Stevenage starting XI: Smith, James-Wildin, Coker, Reeves, Cuthbert, List (Osborne 70'), Taylor, Andrade, Lines (Marshall 58'), Reid (Norris 63'), Melbourne.
Unused subs: Anang, Osborne, Daly, Read, Barry.
Colchester United starting XI: George (Turner 48'), Chambers, Skuse, Nouble (McCoulsky 79'), Sears, Chilvers, Jasper, Eastman (Smith 79'), Dobra, Tchamadeu, Daniels.
Unused Subs: Welch-Hayes, Hannant, Coxe, Cooper.