Having to dig in at the back in the opening 45 minutes, Nathan Baxter produced a couple of fine early saves before being finally beaten shortly before the half-hour mark.
Tom Eastman, up from the back, converted from a corner to net the difference at the break despite an even share of possession.
After starting the second half brightly, Alex Fisher had Town’s best chance of the game but the U’s doubled their advantage through an unstoppable Frank Nouble strike.
With time at a premium the green and whites searched for a way back into the Sky Bet League Two encounter but with numbers forward conceded a second Nouble goal late on, before Tom James netted a stunning free-kick with the last kick of the game for nothing more than a consolaiton.
Facing a second away trip in the space of a few days, Darren Way opted to make three changes from the side that suffered a first defeat on the road in four at Crawley.
Making a change in each department, Bevis Mugabi came in for Enes Mahmutovic at the heart of defence whilst Yoann Arquin replaced Jake Gray on the wing.
Jordan Green dropped back into midfielder to make room for Fisher who netted the winner against the U’s last season, meaning Rhys Browne dropped to the bench.
Unable to call upon eight players due to injury, Town were at least boosted by Alex Pattison who shook off an injury picked up at the weekend to start in Essex.
On a cold night at the JobServe Community Stadium it was the hosts who were quickest out of the blocks, enjoying an early corner which almost brought the opener.
Half clearing their lines, Town could only tee up Sammie Szmodics who saw his curling strike from just inside the box palmed away by a diving Baxter.
The Chelsea loanee was the visitors’ busiest player in the opening 10 minutes, called into action once again to push Ryan Jackson’s well-hit drive behind for another corner.
Avoiding a disastrous start, Olufela Olomola had a couple of tame efforts at the other end but the U’s remained a threat in front of their home supporters.
Already threatening from corners the hosts eventually made one count, as Pell’s header only fell to Eastman who poked home past a hapless Baxter.
Not to be deflated by falling behind the Glovers responded with their best spell of the half, producing some neat football under the lights.
Fizzing the ball around purposefully, Jordan Green and Olomola looked to cause havoc in the final third but faced with a wall of defence, were unable to engineer a chance of worth.
Patiently playing out from the back the tactic almost backfired on 42 minutes when Shaun Donnellan’s back-pass didn’t have the power needed to reach Baxter.
Colchester’s top scorer Luke Norris senses an opportunity but Town’s stopper spread himself well to block the shot and allow Bevis Mugabi the chance to clear from a yard out to keep the score 1-0 at the halfway stage.
Not wanting the trip across the country to be in vain Way’s side came out reinvigorated and almost leveled three minutes after the restart.
Green’s lightning pace saw him race down the right-hand side before firing in a low cross for Fisher whose first-time strike whizzed over the bar.
Sensing that Town were building momentum Colchester responded with a chance of their own in a bid to build a cushion.
Senior and Norris linked up well to allow the latter to unleash a vicious looking strike which flew just over as the game opened up and became end-to-end.
With the tempo increasing the next goal became of increasing importance and just before the hour-mark it came.
Winning possession on the halfway line, Nouble drove at the Town back four and with the hint of a deflection, cannoned a strike up and over Baxter to make it 2-0.
Needing a big turnaround both Jake Gray and Omari Patrick were introduced and the duo combined well to allow Olomola some space in the box, only for the loanee’s strike to be lashed wide.
With little to lose the Glovers went for broke in the final 10 minutes as Rhys Browne came on for captain Sessi D’Almeida to give the visitors four attacking outlets.
However, the green and whites were dealt a further blow inside the final 10 minutes when Nouble competently rifled in his second of the night to round off the hosts’ scoring.
Deep into stoppage time James netted an unstoppable 25-yard free-kick to make it 3-1, but it wasn’t enough to stop Yeovil suffered back-to-back defeats for the first time in 2018/19.
Colchester United: Barnes; Jackson, Eastman, Prosser, Vincent-Young; Pell, Lapslie (Wright 88′); Senior (Dickenson 81′), Szmodics, Nouble; Norris (Mandron 89′).
Subs not used: Gilmartin, Comley, Kensdale, Collins.
Yeovil Town: Baxter; James, Mugabi, Donnellan, Dickinson; Green, D’Almeida (Browne 79′), Pattison, Arquin (Patrick 68′); Olomola, Fisher (Gray 60′).
Bookings: Dickinson 52′
Subs not used: Phillips, Mahmutovic, Clarke, Rogers, Browne.