Danny East and Anthony Charles have joined Northampton's list of absentees.
On-loan right-back East is out of the second round tie after suffering a shoulder injury during the 3-1 defeat at Bristol Rovers on Saturday. It is hoped the Hull youngster will be fit for the League Two game against Exeter at Sixfields at the weekend.
Centre-half Charles will serve a one-match suspension following his red card against the Pirates.
Fellow defenders John Johnson (knee) and Seth Nana Ofori-Twumasi (knee) and central midfielders Luke Guttridge (foot) and Ben Harding (ankle) remain unavailable.
"The squad is looking thin but we've got to get through it and fulfil these games," said Town boss Aidy Boothroyd, who hopes to complete a loan signing in time for Tuesday night's game.
"We have to persist, get our heads down and get on with it."
Joe Dunne is set to freshen up his starting XI for Colchester's trip.
Dunne has rightly kept faith with an unchanged line-up for his first three games in charge, clocking up the U's first wins of the season against Hartlepool and Swindon before being brought back down to earth by Yeovil on Saturday.
And that is set to hand Dunne, who is not expected to have any fresh injury or suspension concerns, the chance to rotate and take his first look at a number of players from the off.
Ian Henderson, Clinton Morrison, Freddie Sears, Karl Duguid, Matt Heath and John White all come into contention for starting berths at Sixfields as Colchester aim to make the southern section quarter-finals.
