The Spireites fell out of the play-off places after Shrewsbury Town scored in stoppage time to secure a 3-2 win.
Trey Ogunsuyi’s double had cancelled out Tom Naylor’s opener before Tom Pearce looked to have earned Chesterfield a late point with his fantastic free-kick.
However, deep into time adde on, Shrewsbury secured all three points as Sam Stubbs looped his header into the far corner.
The defeat sees Chesterfield drop down to eighth, one place outside the play-off picture with 10 games remaining.
Paul Cook named an unchanged side from the one that convincingly beat Colchester United 3-0 on Tuesday evening, as Malik Owolabi-Belewu retained his place in the side after an impressive debut for the Spireites.
It was a fast start by both sides, and the first save of the afternoon fell to Zach Hemming, who did well to parry Will Boyle’s long-range effort.
With 20 minutes on the clock, Dilan Markanday took aim from distance, but Matthew Cox was equal to the effort.
Chesterfield maintained the pressure as Will Grigg saw his header go narrowly over moments later.
Then, as the chances continued to come, Ryan Stirk had a sight of goal, but his low effort was pushed to safety.
And finally, in the 31st minute, Chesterfield took a deserved lead.
Naylor was found in the box by Markanday, and the midfielder expertly curled his strike into the bottom corner.
The visitors had an opportunity to level immediately after when Ismeal Kabia’s cross was headed over from five yards by Tom Anderson.
James Berry then had two great opportunities, both of which Cox thwarted.
First, his stinging volley was palmed away before testing the keeper again with an effort following a mazy run into the area.
Despite Chesterfield dominating large parts of the game, they were pegged back in stoppage time of the first half.
Tom Sang’s deep free-kick was powerfully headed home by Ogunsuyi to make it 1-1.
Chesterfield started the second period on the front foot as Markanday and Berry both had chances to restore the lead.
Just after the hour mark, the home side again had a great opportunity to get a second, but Grigg couldn’t turn Markanday’s cross goalwards.
But again, despite Chesterfield’s dominance, Shrewsbury took a 70th-minute lead.
Hemming was pressed by Ogunsuyi, and the forward poked the ball from under the goalkeeper’s foot before he had a chance to kick it clear.
Tom Pearce had a powerful effort from distance with 10 minutes left to play, but Cox watched as it flew over the bar.
Pearce again was involved with five minutes to play, and this time the left-back found the back of the net with his strike.
His fiercely struck free-kick from 25 yards left Cox with no chance as the Spireites got back on level terms.
Armando Dobra had the SMH Group Stadium on its feet late on as they thought he had scored, but his effort had just nestled in the side netting.
And deep into stoppage time, Shrewsbury dealt the final hammer blow as they retook the lead and secured all three points.
Sang delivered another deep free-kick, and with a lofted header, Stubb’s found the far corner, which sent the away fans into raptures.
The Spireites couldn’t find a response as they dropped down to eighth.
Chesterfield: Hemming, Curtis, McFadzean, Swinkels, Owolabi-Belewu (Pearce 71), Naylor (Mandeville 71), Stirk (Dobra 46), Braybrooke, Markanday, Berry (Dickson 78), Grigg (Bonis 71)
Attendance: 8,118
