A James Norwood header two minutes from time gave Rovers a precious three points at Eastleigh.
Tranmere made a couple of changes to the side which started last weekend with Lois Maynard and Jake Kirby coming into the starting eleven.
The first chance on goal fell to Rovers on 12 minutes as a corner from the right found its way to Jake Kirby who turned and fired a low shot which Flitney saved low to his left.
Lois Maynard had another opportunity on 21 minutes as a ball by Steven Jennings found him racing on goal but the keeper came out and cleared his lines.
James Norwood won a free kick on 27 minutes and the 20-goal striker picked himself up and fired a low effort which Ross Flitney saved low down.
It remained goalless to half time and Steve McNulty was replaced by Martin Riley at the break.
Kirby was close to getting on the end of a forward pass from Jennings at the start of the second period.
Adam Mekki was introduced on the hour mark, replacing Kirby.
On 67 minutes, Tranmere had the ball in the net as Norwood took the ball down and volleyed into the top corner but the offside flag was raised and the goal was disallowed.
Five minutes later, Mekki drilled in a powerful shot which Flitney saved before getting up and blocking Norwood's rebound.
Eastleigh's first meaningful effort on goal came on 81 minutes when a right wing cross by Joe Partington was headed by Jai Reason but superbly saved by Scott Davies.
Norwood scored for Rovers on 88 minutes when a free kick was played into the box, flicked on by Lois Maynard and Norwood nodded over Flitney to give Tranmere the lead.
Eastleigh had a couple of shots over the bar in the dying embers but Tranmere stood strong to claim the three points.
Eastleigh: Flitney, Partington, Green, Evans, Turley, Reason, Midson (Odubade 68), Strevens, Drury (Cook 57), Coulson (Payne 18), Tubbs. Substitutes: Poke, Reid.
Tranmere: Davies, Vaughan, McNulty, Ihiekwe, Ridehalgh, Maynard, Jennings, Harris (Taylor-Fletcher 82), Hughes, Norwood, Kirby (Mekki 60). Substitutes: Turner, Higdon.
Referee: Ollie Yates
Goals: Norwood (88)
Attendance: 3269 (533)