Blues comprehensively beaten against the Spireites in Sky Bet League 1.
A brace from experienced forward Sylvan Ebanks-Blake helped Chesterfield condemn Southend United to a 3-0 defeat in Derbyshire.
The striker scored his first mid-way through the first half after fellow frontman Lee Novak had given the Spireites an early lead, before Ebanks-Blake added his second and Chesterfield's third of the night from close range just after the hour mark.
Manager Phil Brown named an unchanged XI and substitutes for the game against Dean Saunders' side after a good win against Barnsley at Roots Hall just three days earlier.
Despite being back available following his suspension, Gary Deegan missed out on the squad, but did travel, as did David Worrall, Adam Thompson and Luke O'Neill.
It was an encouraging start for the Blues in the early stages before the hosts took the lead through striker Novak.
The Birmingham City loanee was put through and pushed the ball to the right of the onrushing Dan Bentley before slotting home.
Dean Saunders' men smelt bloody and has a succession of corners in the space of just a couple of minutes, with Ryan Leonard heading over his own goal before Ebanks-Blake headed over from a Danny Gardner flag kick.
With the game close to being 20 minutes old, Sam Morsy fired narrowly over the upright from long range before fellow midfielder Jay O'Shea saw his strike fly inches past Bentley's left-hand post.
For the visitors, Leonard hit a couple of efforts wide, whilst Joe Pigott battled to get away from his marker before seeing his shot blocked in a frantic opening half an hour.
On 32 minutes, a neat corner routine found Morsy again, and his effort went through a couple of bodies before forcing Bentley into a save.
Moments later, Anthony Wordsworth fired over the bar after neat play involving the marauding Ben Coker and Pigott.
And it was 2-0 seven minutes before half time, this time it was Ebanks-Blake the scorer.
The tricky Gardner cut in from the left before unleashing a strike which was cleared off the line by Coker, falling to Ebanks-Blake who in turn smashed high into the net from close range.
With the half coming to an end, David Mooney saw a chance headed off the line, before Anthony Wordsworth saw his volley end up in the gloves of goalkeeper Tommy Lee to give the visiting Southend supporters hope of a resurgence in the second 45.
It was better after the break from the Blues, and eight minutes into it, Pigott was played through and poked goalwards, with the ball beating Lee but also beating the post by a matter of inches.
However, just after the hour Chesterfield grabbed their third of the night and it came from the boot of Ebanks-Blake again.
The striker prodded home from inside the six-yard box after a corner wasn't cleared to make it a brace for the former Wolves frontman.
Straight after the goal, Brown threw on Jack Payne in place of Wordsworth before Mooney was replaced by Chesterfield-born Kevan Hurst soon after.
With 11 minutes remaining Brown made his final change, taking off Pigott for Jason Williams.
Soon after and Bentley had to react smartly to tip over a curling Novak effort from an acute angle.
Up the other end, Blues came close to grabbing a late consolation when skipper John White headed a Hurst cross narrowly over the bar, but the Blues' three-game unbeaten run came to an end at the Proact Arena.
Blues: Bentley; White (c), Bolger, Prosser, Coker; Wordsworth (Payne, 64), Leonard, Timlin; Mooney (Hurst, 70), Pigott (Williams, 79), Weston
Unused substitutes: T. Smith, Atkinson, McLaughlin, Rea.