Wednesday had a miserable night after Sunday's win over Sheffield United and had defender Tony McMahon sent off.
Wednesday were still basking in the warm glow of their victory over Steel City rivals United just two days earlier when Barnsley caught them cold.
Martin Devaney's dazzling sprint down the left had Wednesday back pedaling and when he swung in a testing low cross Lewis Buxton struggled to clear.
Jon Macken snapped up the ball and picked out strike-partner Iain Hume, who fired in an accurate shot from 15 yards out to claim his fourth goal of the season.
Barnsley, who are floundering in the relegation zone, threatened to stretch their lead with a string of chances.
Devaney cut in from the right and lashed a low shot that went close before Jamal Campbell-Ryce lashed another effort just wide.
And when Macken slid a crisp pass for Devaney to sprint into the area, he blew his chance and Lee Grant saved well.
Campbell-Ryce picked out Hume with a right-flank cross but the Canada international's tame header floated wide.
But 11 minutes before the break Wednesday were down to ten men when on-loan McMahon was sent off for a two-footed tackle on Rob Kozluk.
Barnsley's second came with a 61st minute penalty from Campbell-Ryce after Mark Beevers grappled with Stephen Foster.
Wednesday hit back from the spot eight minutes later when sub Leon Clarke converted with his first touch after being brought down.