The deadlock was broken with a little under 10 minutes left to play in the first-half, when Tarique Fosu slammed an effort home from range in an opening 45 minutes that had shown signs of encouragement for the Glovers.
The visitors threatened to get back into the game after the break before Fosu doubled his tally on 77 minutes, punishing a defensive mix-up in what was an otherwise solid display.
Defeat confirmed a 20th place finish for the Green and Whites, despite boasting their highest points tally for the last four seasons.
Darren Way made one change to the side that secured league status with a 1-1 draw against Stevenage seven days ago, with Francois Zoko missing out with a back injury.
Tottenham loanee Shayon Harrison replaced Town’s top scorer to partner Jean-Louis Akpa Akpro upfront, whilst 17-year-old Academy full-back Joe Tomlinson took a spot amongst the substitutes for his first involvement with the first-team.
Keen to send the travelling faithful home with three points and derail Colchester’s hopes of sneaking a spot in the play-offs the Glovers started brightly, looking lively in the final third.
Neat link up between Akpa-Akpro and Ben Whitfield released the latter down the right, with the Bournemouth’s loanee pull-back finding Otis Khan whose first-time effort flew just over the bar.
The visitors continued to enjoy an even share of possession in the opening exchanges, with the U’s taking 20 minutes to register their first shot, which Drey Wright dragged wide.
That effort did spur the hosts on however, as John McGreal’s side threatened again five minutes later when Matthew Briggs went close to heading home Richard Brindley’s cross.
With a sense of déjà vu, the same combination endangered Yeovil’s goal once again just past the half-hour mark, with the former Fulham defender this time heading into the arms of a thankful Jonny Maddison.
Whilst Town were doing a good job of containing the Essex outfit, there was increasing pressure being inflicted by Colchester which paid off as the game ticked into the 36th minute.
Loanee Fosu engineered space 25 yards from goal, before dispatching a low drive into the bottom corner giving the U’s the lead with their first meaningful attempt on target.
In fact, the opener was as good as it got in terms of goal scoring opportunities before the break, as Town quickly regrouped to head into half-time in the ascendancy.
Knowing they were likely to require more goals to claim their play-off place, Colchester were quick out of the blocks after the restart with Wright blazing over the bar when through on goal six minutes into the half.
As the hour-mark approached Brindley saw a dipping free-kick from 35 yards drop just over Maddison’s crossbar, before Yeovil began to threaten at the other end.
The energetic Whitfield and lively Khan started seeing more of the ball, before Nathan Smith headed straight at Sam Walker from a Matty Dolan corner.
With the clock reading 73 minutes Whitfield had the chance to level the scores running on to substitute Tom Eaves’ pull-back, but the midfielder couldn’t find the target as the Green and Whites threatened to ruin the U’s play-off hopes.
But the Glovers were made to rue that missed opportunity as the game ticked towards the final 10 minutes when Fosu netted a second with an emphatic finish from inside the box.
Both Brandon Goodship and Matt Butcher were introduced as Way made all three substitutions in a bid to get back into the game and give the 231 travelling supporters something to cheer, but Walker wasn’t called into action again.
The referee’s full-time whistle saw the season end in defeat, leaving Way to reflect on a campaign of fine lines.
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Colchester United: Walker; Eastman, Wynter, Elokobi; Brindley, Lapslie (Loft 82’), Fosu, Wright (O’Sullivan 88’), Murray, Briggs; Porter (Bonne 86’).
Bookings: Lapslie 27’
Subs not used: Brill, Kinsella, James, Pyke.
Yeovil Town: Maddison; Shephard, Lacey, Smith, Dickson; Whitfield (Goodship 83’), Lawless (Butcher 81’), Dolan, Khan; Akpa Akpro, Harrison (Eaves 68’).
Bookings: Lawless 24’
Subs not used: Mugabi, James, Tomlinson, Sowunmi.