The Spireites were edged out 2-1 at Torquay United after the hosts struck a stoppage-time winner.
Tom Denton had cancelled out an early opener for the hosts, scoring the equaliser just before the break, but there was late heartbreak for the Spireites when Asa Hall scored in time added on.
John Pemberton made one change to the side that beat Woking 4-0 at the weekend with Milan Butterfield replaced by Liam Mandeville.
Chesterfield made a bright start to the game, pressuring the Torquay back-line.
Mandeville latched on to Denton’s knock down but he couldn’t get the shot away.
The home side took the lead in the ninth minute when Ben Whitfield’s low cross ricocheted off Kyle Letheren and into the path of Louis Britton, who tapped the ball into an empty net.
Denton came very close to levelling the scores, latching on to a cross but his header hit a post.
Torquay looked to double their advantage and went close twice through Whitfield.
Letheren was equal to one of the efforts and the other went just wide.
The Spireites scored an equaliser in the 43rd minute through Denton. Mandeville’s low free-kick was palmed out to the striker, who lashed the rebound into the bottom corner.
The second half began with Torquay doing the pressing. Whitfield latched onto a corner, but saw his header go wide of a post.
Just after the hour-mark, Chesterfield found themselves lucky not to go behind. Britton rose highest from a corner, but his header was cleared off the line.
The Spireites’ first attempt of the second half came with ten minutes to go when substitute Luke Rawson looked to chip the keeper from range, however his effort rolled wide of the post.
It was the home side who grabbed a last-gasp winner when Jake Andrew’s corner was flicked past Letheren into the bottom corner by Hall.
Chesterfield now look ahead to Saturday when they face Stockport County at the Technique Stadium in a match being televised live on BT Sport.
Chesterfield: Letheren, Cropper, Sharman, Evans, Maguire, Buchanan, Weston, Smith, Mandeville (Butterfield 78), Kiwomya (Rawson 53), Denton (McKay 68).
Unused substitutes: Wharton & Hollis.