It's defeat on the road for the Reds as a failure to take chances is punished accordingly by in-form Blackburn.
Valérien Ismaël had made two changes to his starting XI with Matty James and Dominik Frieser missing out for Herbie Kane and Luke Thomas respectively.
There was an early Tom Trybull effort from range for Barnsley to deal with, as the German's powerful drive was only parried by Jack Walton but Mads Andersen was on hand to hook the rebound clear.
The Reds settled and went on to dominate the first half but spurned a number of presentable opportunities.
Kane saw a 30-yard free-kick plucked out of the top corner by Rovers' 'keeper Thomas Kaminski, before Kane's inswinging corner was headed over from six yards by Andersen under a challenge.
Conor Chaplin saw a half-volley fire into the ground and take a deflection, seeing it go an inch wide from just inside the Rovers box.
The diminuitive striker then shot straight at the hosts' goalkeeper after great work by Callum Styles wide left.
Another set piece from the right by Barnsley saw Cauley Woodrow get a header badly wrong, as the visitors failed to capitalise on their pressure.
Walton had to be sharp to save from Ben Brereton with his feet as we approached the break, before Rovers punished the Reds just two minutes later.
Andersen was muscled out of it on the left by Ryan Nyambe and the full-back's low cross was emphatically crashed home from 12 yards by ex-Reds loanee Adam Armstrong.
HALF-TIME: Rovers 1-0 Reds
Following the break, the Reds had a 15 to 20 minute spell where they looked close to levelling proceedings with Woodrow seeing a goalbound strike blocked inside the box, then Styles firing well over soon after.
Callum Brittain was impressive down the right but it was through the middle where he excelled on the hour-mark as he somehow glided beyond defenders before curling an effort wide from the edge of the box, as substitute Victor Adeboyejo screamed for a pass.
Andersen came the closest for Barnsley with a header that was wonderfully tipped behind by Kaminski, following a delicious Kane corner kick.
But the Reds were made to pay for their profligacy in front of goal on 77 minutes as Nyambe again beat his man too easily before his cleared cross dropped for Sam Gallagher to stroke home from the edge of the box for 2-0.
Romal Palmer - off the bench - did make an impact, as his goal with five minutes of added time remaining gave the Reds brief hope of a turnaround, but it wasn't to be.
FULL-TIME: Rovers 2-1 Reds
Walton, Brittain, Sollbauer, Helik, Andersen, Styles (Palmer), Mowatt (Oduor), Kane, Thomas (Frieser), Chaplin (Adeboyejo), Woodrow (Miller).
Unused: Collins, Moon, James, Schmidt.