Despite turning in an impressive display and coming within a whisker of a shock win, a point was never enough to save them.
After they grabbed an early lead, Barnsley quickly levelled and then saw a string of chances go begging before Posh went ahead again.
But Barnsley equalised with six minutes to go.
Posh, who lost narrowly to Newcastle at the weekend, swept in front at the first attempt after just five minutes to rock the home fans.
Dominic Green's free-kick from the left was headed down by Exodus Geohaghon and, as the Barnsley defence dithered, Ryan Bennett stole in and prodded the ball in from close range.
Barnsley, who held local rivals Sheffield United to a goalless draw on Saturday, were level a minute later.
Martin Devaney sent in an accurate cross from the right that skidded through to Iain Hume and the Canada international clipped in his shot off the post from 10 yards.
Devaney sent a 40-yard cross-field pass for Adam Hammill on the right and the former Liverpool winger skipped into the box and picked out Jon Macken with a clever chip, but Macken's strong header brought a super reaction stop from James McKeown.
Then McKeown produced another spectacular stop to deny Barnsley when he beat away Stephen Foster's thundering header from Hume's corner.
Four minutes later Posh survived again when Macken stabbed the ball past the keeper and hit the post and somehow Hammill managed to fire wide from 10 yards.
A Hugo Colace shot through a crowd was blocked by Gabriel Zakuani and another Hammill effort was well saved
When Reuben Reid showed slick skill to weave his way into the Barnsley area, Steele had to drop smartly to block his shot.
Steele then had to stretch to turn away a shot from Craig Mackail-Smith.
Peterborough finally regained the lead after 77 minutes as Barnsley were caught out at the back and the ball dropped into the path of Tommy Rowe, who drove in his angle shot.
Then with six minutes to go, Hume ghosted in again to plunder his second with a simple finish after Andy Gray's header went over McKeown.