Nine-goal top-scorer Simeon Jackson and midfielder Danny Jackman are ruled out, with Jackson struggling with a hamstring injury and Jackman dislocating his shoulder at Southampton on Saturday.
Defender Josh Gowling is banned after collecting his fifth booking of the season at St Mary's while Simon King (ankle ligaments) is a long-term absentee.
Striker Mark McCammon (hamstring) and Garry Richards (broken toe) returned to the bench at Southampton and may be recalled to the starting line-up.
Midfielder Stuart Lewis (ankle ligaments) is back in training but is unlikely to feature.
Gillingham, who defeated Colchester on penalties in the previous round, are aiming to reach the third round of the Johnstone Paint Trophy for the second time in three seasons.