Having ended a 78-year hoodoo by winning at Oakwell on Boxing Day, the Clarets rediscovered that old habits die hard as goals from Jacob Butterfield and Ricardo Vaz Te condemned them to back-to-back Championship defeats.
Rarely a happy hunting ground, Burnley again left South Yorkshire empty-handed as they made an unwanted start to a spell of three away games in eight days.
Ross Wallace saw a fourth-minute effort cleared off the line and Jay Rodriguez rattled the woodwork with the score at 1-0.
But the Clarets were again far from watertight at the back and a tidy Barnsley side was good enough to make enough of their chances count and leave Burnley treading water in the Championship table.
Boss Eddie Howe had given a vote of confidence to the side that lost to Reading last Saturday by giving the same players the chance to make amends for their late lapse against the Royals.
However, there was a change to the Clarets' formation with Rodriguez initially taking up a wider position on the left with Wallace moving into a central attacking midfield position.
Jacob Butterfield puts Barnsley ahead
And Wallace was desperately close to giving Burnley an early lead.
Keiran Trippier, back at the club where he spent last season on loan, was quick to join the attack and when his cross was only half cleared, Wallace connected with a thunderbolt from the edge of the penalty area which was blocked on the line by defender Rob Edwards.
The Clarets looked a threat going forward but Barnsley were just as incisive with an attack well led by former Burnley striker Andy Gray.
Jimmy McNulty headed a Butterfield corner just wide after 19 minutes and two minutes later the home side went ahead.
Butterfield and wideman Jim O'Brien caused Burnley plenty of problems and those two played a key role in the goal.
Junior Stanislas tries to get the Clarets moving
Trippier had already won a couple of vital challenges against Craig Davies at the far post but when Davies managed to meet an O'Brien cross with a downward header, the outstanding Butterfield pounced to poke the ball home from six yards.
The Clarets' response was almost instant with Rodriguez, another former Barnsley loanee in the Burnley ranks, rattling the crossbar with a dipping drive.
But Barnsley were not prepared to sit on their lead and five minutes before the break almost went 2-0 up.
Danny Drinkwater's fiercely struck free-kick was only parried by Lee Grant and it took the combined efforts of Grant and David Edgar to block the follow-up as Edwards and Gray both closed in on the loose ball.
Lee Grant takes a painful blow
With Wallace restored to a wide position and Rodriguez back up front in a 4-4-2 formation, the Clarets looked for a route back into the game and two minutes into the second half they almost found it as Charlie Austin saw an effort blocked by McNulty before firing into the side netting.
But in a breathless spell Barnsley also had opportunities and could have put the game beyond the Clarets.
Either side of a swerving Butterfield drive which Grant did well to save, Davies twice had clear-cut chances.
The first saw him faced with just Grant to beat with a clear run on goal but his hurried finish let the Clarets off the hook and then when he again got behind the defence from a Gray flick, the former Chesterfield striker again hooked his effort wide.
In a wide open game, Wallace countered with a long-range effort which Luke Steele saved at full stretch before the Barnsley keeper was almost beaten by a remarkable effort from Chris McCann.
Luke Steele breathes a sigh of relief as Chris McCann goes close
Steele's dash from his box thwarted Marvin Bartley but when his clearance was collected by McCann, the Clarets' skipper took aim at the empty target from almost 50 yards out and was only inches wide as Steele threw himself desperately across goal.
The Clarets were gradually cranking up the pressure but with nine minutes to go they were caught out on the break as Barnsley nicked a second goal to seal the points.
Scott Wiseman's hurried ball up the line eluded Edgar and when the ever-willing Gray picked out substitute Vaz Te, the former Bolton striker delivered an assured finish inside Grant's near post to bank the points.
Burnley: Grant, Trippier, Easton, Edgar, Amougou, Bartley, McCann, Wallace (Hines 88), Rodriguez, Austin, Stanislas (Treacy 68). Substitutes: Mee, Marney, Stewart.
Barnsley: Steele, Wiseman, Edwards, Foster, McNulty, O'Brien, Davies (Vaz Te 75), Doyle, Butterfield, Drinkwater, Gray. Substitutes: Addison, Park, Stone, Preece.
Jay Rodriguez can't find a way through