The Reds, who were beaten 5-0 by Manchester City in the FA Cup on Saturday, came into the clash without a win in three league games and sitting bottom of the table.
But Jason Scotland gave them an early lead when he struck home following some powerful forward play, and they then showed their mettle as they regained the lead, through Chris Dagnall's penalty, just five minutes after Leonardo Ulloa had headed Brighton level on the hour mark.
Ulloa's night ended in misery, though, as he saw a late penalty saved by heroic Barnsley goalkeeper Luke Steele as the hosts held on for a valuable win which lifts them out of the bottom three.
For Brighton, who came up against formidable opposition in the shape of Steele, it is now three games without a win and they remain a point out of the top six.
Will Buckley should have put Brighton in front in just the third minute, but he shot off target after Scott Wiseman's poor clearance fell invitingly to him on the edge of the area.
Scotland dragged a 20-yard effort wide for the hosts, but he soon made up for that miss by putting his side ahead in the 15th minute. Jim O'Brien fed a ball forward and the former Swansea striker shrugged off Adam El-Abd's challenge to power home from 12 yards.
Gus Poyet's play-off chasers finally got going in the second half, testing Steele for the first time, with the Reds keeper first getting down well to deny David Lopez and then coming out to block Andrea Orlandi's effort.
Steele's heroics proved to be in vain, though, as just before the hour Brighton levelled. Inigo Calderon picked up the ball on the left and whipped in a vicious centre that an unmarked Ulloa headed past Steele before he could move.
Barnsley were not rattled, though, and restored their lead just five minutes later, courtesy of Dagnall's accurate penalty, which came after the cumbersome El-Abd had pulled at the striker's shirt from O'Brien's cross.
Brighton thought they had their reward for a spell of pressure with 10 minutes to go when Heywood pointed to the spot for a second time following Perkins' foul on Calderon, but Steele provided more resistance by palming away Ulloa's penalty.
Brighton poured everything forward in search of an equaliser but Barnsley held on.