The Shrimpers striker was making his first appearance since picking up a hamstring problem in the 1-1 draw at Leyton Orient in mid-January.
But he lasted just 56 minutes on his come-back and is now facing another spell on the sidelines.
Blues boss Steve Tilson said: "It's too early to tell how serious the injury is but it's in the same area again so it doesn't look good for James unfortunately. I would be very surprised if he was fit again in time for Saturday and it's disappointing because we have real injury problems in attack at the moment."
Blues fielded three trialists against their Essex rivals - Charlton Athletic's Sam Long, Norwich City's Robert Eagle and Ipswich Town's Ed Upson.
But Colchester opened the scoring through Russell Malton in the 37th minute before Medy Elito and Jordan Pavett both scored in the final 15 minutes.
Southend: Joyce, Harvey (Forshaw 72), Jones (Osifuwa 82), Whittaker, Herd, Long, O'Keefe, Upson, Eagle, Ljungstrom, Walker (Crawford 60)