Jermaine Easter's 54th minute strike - his first since arriving on loan from Plymouth - proved enough to secure the U's victory in a dour contest at the Weston Homes Community Stadium.
Easter fired in a spectacular half-volley from close range, after Clive Platt had headed down David Perkins' diagonal pass.
Colchester had made most of the running in the first half, with Easter going close with a fierce shot which Yeovil keeper Josh Wagenaar tipped around the post.
But Yeovil threatened on the counter-attack and Aidan Downes wasted a good chance just before half-time when he shot weakly at keeper Dean Gerken.
Easter broke the deadlock early in the second half and although Luke Rodgers and Andy Welsh went close for the Glovers late on, Colchester held on for a crucial three points.