A brace by debutant striker Paddy Madden and a James Hayter strike saw off the resurgent U's.
The home side took the lead in the 21st minute when Madden, who finalised a one-month loan deal from Carlisle United on Thursday, pounced after goalkeeper Mark Cousins failed to hold Hayter's initial effort.
Colchester hit back four minutes later when Sanchez Watt's teasing cross found Gavin Massey, who knocked the ball into the direction of the lurking Jabo Ibehre to tuck away his third goal in as many games.
Yeovil, who saw manager Gary Johnson linked with the vacancy at Bournemouth this week, restored their advantage after 33 minutes when Madden fired a right-footed shot past Cousins.
In the second half, Korey Smith and Brian Wilson both hit the crossbar before Madden was twice denied a hat-trick by Cousins.
The Glovers secured the win when captain Jamie McAllister's low cross was coolly converted by Hayter 10 minutes from time.