Striker Iain Hulme inspired Tranmere to victory but they will miss out on a play-off position.
The Merseysiders extended their unbeaten run to nine matches and could easily have won by a greater margin.
The deadlock was not broken until the 62nd minute when Hulme volleyed home his 13th goal of the season from a headed pass by Graham Allen.
Hulme had been unlucky with an early effort which was well saved by keeper Ross Turnbull.
The striker turned creator late on, providing the perfect pass for substitute Ryan Taylor to strike with an angled shot in the 88th minute.
Danny Harrison wasted two terrific chances for Tranmere in the first half and Ian Sharps and Gary Jones flashed headers just over.
David Beresford made an impact when going on as a second-half substitute. He quickly created a headed opportunity for Paul Hall which was wasted and then had a shot beaten out by Turnbull.
Home keeper John Achterberg did well to make a blocking save from hard working Gary Birch in the 19th minute.
Birch later headed wide with one effort, saw another deflected for a corner and a third pass inches wide of a post.
Barnsley were not helped by an early injury to Steve Hayward who was forced off in the 25th minute with a dead leg.
Skipper Craig Ireland was stretchered off in the 70th minute but manager Paul Hart said there was fortunately no serious injury.
"He has just got a bit of a headache," said Hart.
Kevin Betsy and Chris Shuker did well for the Reds but it was a tenth away defeat.
"I thought we were well in the game in the first half, but we did not put away our chances. Physically we went under in the second half when we looked weak.
"I did not look at it as a non-incentive match and I hope the players did not.
Sixteen of them are playing for new contracts." Tranmere manager Brian Little described it as "an okay sort of game with an ingredient missing".
"I thought whoever scored first would win and fortunately it was us.
"Hulme did very well. He always looked dangerous and caused Barnsley many problems."