Adam McGurk's first half goal wasn't enough for Rovers as they conceded late on to lose 2-1 at Yeovil Town.
The 22-year old scored his third goal of the season early on only for the Glovers to level midway through the first half.
In a closely contested match Robbie Weir went close to scoring a winner when his shot crashed against in the second half but Yeovil claimed all three points when Andrew Williams headed in on 95 minutes.
There were three changes for Rovers from the 2-2 draw against Brentford, with Ian Goodison returning the starting line-up, a change that meant Ash Taylor pushed forward into midfield. Robbie Weir came in for Joss Labadie while Adam McGurk took the place of Martin Devaney.
The opening stages were dominated by Yeovil who were looking to make a bright start under new manager Gary Johnson. Zoumana Bakayogo did well to clear the danger on two minutes while Andrew Williams fired wide from a long ball from Ryan Dickson on three minutes.
Rovers responded well with Mustafa Tiryaki winning a corner from which Lucas Akins sent a looping header just over the bar after Ash Taylor missed with his attempt.
On nine minutes a foul by Bondz N'Gala on Tiryaki earned the Yeovil defender a yellow card and Rovers a free kick. McGurk whipped in the set piece which went over the wall and into the corner of Rene Gilmartin's goal to give Tranmere an early lead.
Dickson shot over the bar from 12 yards for Yeovil after appeals for a foul by Paul Rachubka were turned down while another McGurk free kick was turned just beyond the path of Goodison at the far post by a Glovers defender soon after.
The home side levelled on 19 minutes. A corner from Nathan Jones was cleared back as far as the former assistant manager, and though his ball back into the Rovers area was headed away from goal by Goodison, Joe Edwards fired a shot through a crowd of players and past Rachubka into the net on his Yeovil debut.
McGurk sent a long range effort wide of the target on 24 minutes and Gilmartin beat Akins to claim a cross soon after as Rovers looked to re-establish their lead.
Jon Obika headed over the Tranmere bar from Dickson's cross on 35 minutes and sent another effort wide of the target three minutes later.
The final chance of the half saw Gilmartin comfortably save McGurk's free kick at his near post.
Akins and Tiryaki couldn't take advantage of a well flighted cross from McGurk and Yeovil quickly broke on the counter attack and saw Andrew Williams put a shot just past the post.
Spurs loanee Dean Parrett fired wide of Rachubka's goal on 55 minutes while Tiryaki shot narrowly wide after good work from Akins and John Welsh three minutes later.
Rovers' first substitution saw Devaney replace Akins on the right side of midfield while there was a break in play soon after due to an injury to referee Simon Hooper who was eventually replaced by one of his assistants.
When play resumed Tiryaki headed over from a McGurk corner and soon after Rachubka tipped a shot from Joe Edwards over his bar.
Zoumana Bakayogo broke forward on 76 minutes and sent a powerful low effort wide of the target and Tiryaki couldn't get enough of a touch on a McGurk cross, with the ball heading just past goal.
Rovers were gradually taking control of the game and saw Robbie Weir curl a shot inches off target on 79 minutes.
The midfielder went even closer on 85 minutes when he ran on to a superb pass from Welsh got into the area and saw his attempt hit the post and rebound away from goal.
Andrew Williams almost stole the points for the home side with an angled shot which was kept out by Rachubka on 89 minutes while Tiryaki spotted the keeper off his line at the other end in stoppage time and tried his luck from 35 yards only for Gilmartin to save.
With seven minutes of time added on there was still an opportunity for both teams to grab a late winner and it was Yeovil who scored the game's third goal. A cross from Dickson was drilled into the area and Andrew Williams popped up a the near post to head past Rachubka from close range.
Rovers threw men forward in the dying stages in search of an equaliser but they were unable to create another chance on goal.
Yeovil: Gilmartin, N'Gala, Edwards, Ayling, Jones (Gavin Williams 73), Huntingdon, Dickson, Parrett, Upson, Andrew Williams, Obika. Substitutes: Haynes-Brown, Agard, MacLean, Stewart.
Tranmere: Rachubka, Holmes, Goodison, McChrystal, Bakayogo, Welsh, Taylor, Weir, Akins (Devaney 60), Tiryaki, McGurk. Substitutes: Coughlin, Buchanan, Kay, Labadie.
Referee: Simon Hooper
Yellow cards: N'Gala (9), Rachubka (53), McGurk (87)
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