Despite dominating large parts of the match the Bees couldn’t apply the finishing touch to a number of chances as second-half strikes from Jay O’Shea and Neal Togwell stunned Edgar Davids’ side.
Barnet made no changes to the side who beat AFC Wimbledon 1-0 on Monday with goalscoring hero Keanu Marsh-Brown on the bench once more.
The Bees made a bright start to the match and good work from Jon Nurse led to a corner after just two minutes.
Three minutes later the away side ventured forward for the first time and they tested Graham Stack when Togwell’s powerful drive had to be turned round the post.
With 25 minutes played the Bees tested Tommy Lee for the first time but like Stack the Chesterfield keeper was equal to it as he palmed John Oster’s strike to safety.
With six minutes of the half remaining Andy Yiadom burst past Conor Townsend and crossed to Nurse at the near post whose flick was turned behind. From the resulting corner Jake Hyde headed straight at Lee who gathered at the second attempt.
The Bees’ lone striker had another chance before the break but he mistimed his jump and headed wide from a Davids’ free-kick.
Barnet burst out of the blocks in the second period forcing four corners in the space of just three minutes.
Five minutes later Jon Nurse cut into the box and saw his shot deflect wide and from the corner he saw another effort blocked by the Chesterfield backline.
Oster stepped up to take the third corner and Mark Byrne headed towards goal but the away side scrambled it away once more.
Three minutes later Byrne turned provider when his cross found Hyde who flicked the ball wide. However, the offside flag was raised.
Two minutes later the Bees somehow found themselves behind as Jay O’Shea turned on the edge of the box and sent a sensational finish beyond Stack and into the top corner.
With 22 minutes remaining Oster and Davids combined well from a corner to send the Dutchman running into the box. His shot was scuffed but it fell to Tom Flanagan who turned and shot but it was blocked by Hyde who couldn’t get out of the way.
Two minutes later Byrne got in behind the Chesterfield defence with Hyde but the midfielder opted to square to the forward instead of shooting and the ball was cleared behind.
With 12 minutes to play the away side doubled their lead when Tendayi Darikwa turned away from two Barnet players and crossed to Togwell who poked the ball home.
Craig Beattie, Keanu Marsh-Brown and Harry Crawford were all thrown on as the Bees attempted to find a route back into the game but it was too little too late.