Paul Hart's team saw a three-goal lead slip away against Stockport and this time failed to hold onto a two-goal advantage.
Chesterfield showed plenty of spirit to come back but Barnsley will feel they should have closed the game out after Mick Boulding's brace near the end of the first half.
Boulding's pace was too much for Australian defender Aaron Downes who was exposed on both occasions, but Barnsley were less impressive in the face of a strong second-half wind.
Once substitute Caleb Folan reduced the deficit in the 50th minute Chesterfield sensed there was a way back and their pressure was rewarded when Shane Nicholson curled a free-kick into the top right hand corner with two minutes remaining.
Barnsley were unhappy with the performance of referee Mike Dean but they could have taken the points with better finishing.
Barry Conlon wasted a great chance in the 72nd minute when he headed hopelessly wide from only six yards out after Michael Chopra had set him up.
And Boulding could have claimed his hat-trick in the final second of stoppage time but is low shot from just inside the box slipped the wrong side of the post with Carl Muggleton beaten.
But Chesterfield deserved a point for their spirited revival and they have still only been beaten once at home this season.
Barnsley could have become the second team to leave Saltergate with the points but at least their unbeaten run was stretched to eight matches while Chesterfield have now won only once in seven games.