The visitors were unlucky to lose striker Paul Sheilds with a leg injury after only nine minutes and they found themselves behind 60 seconds later.
Tranmere skipper Mike Jackson was given too much space in the box as he headed home from a Gareth Roberts' free-kick.
Gillingham made it 1-1 in the 16th minute. Jackson impeded Gillingham skipper Darren Byfield and the striker found the top left-hand corner with a free-kick from 20 yards.
Byfield was unlucky with another excellent free-kick which hit the crossbar in the 37th minte.
Tranmere, who lost midfielder Danny Harrison with a broken nose midway through the first period, regained the lead 60 seconds after half time.
Chris Greenacre hooked the ball home from Delroy Facey's pass following good work by Sam Aiston.
Byfield rattled the bar again for Gillingham with a 35-yard volley in the 56th minute but he made no mistake from the penalty spot on 63 minutes.
He sent keeper John Achterberg the wrong way from the spot after he had been impeded by Ian Goodison.
Tranmere nearly snatched the points in the closing stages but a Jackson header struck the post.