The Spireites fell to a late defeat as a stoppage-time winner from Brennan Camp gave Eastleigh a 2-1 victory at the Silverlake Stadium.
Paul Cook made six changes after the 3-2 defeat to Dagenham & Redbridge on Tuesday with Ross Fitzsimons, Joe Cook, Tim Akinola, George Cooper, Akwasi Asante and Kabongo Tshimanga coming into the starting line-up.
Tshimanga looked to get the Spireites off to the perfect start, but his powerful effort was saved by Joe McDonnell.
Fitzsimons, who was replacing the injured Lucas Covolan, was called into action early as he denied Tristan Abrahams.
Chesterfield were enjoying the better of the chances as Tshimanga was once again thwarted by McDonnell as the goalkeeper swatted the ball away from goal.
Cooper, who was making his first start since joining in the summer, was giving a good account of himself.
He first almost latched onto a ball from Liam Mandeville, which would have resulted in a certain goal; he then did brilliantly down the left-hand side and set up Mandeville as the midfielder somehow saw his shot roll across the face of goal.
The home side were also creating chances and came close to taking the lead on the half-hour mark as Charlie Carter hit the bar with a header.
The Spireites threatened again soon after as Tshimanga did well to pull the back for Cooper in the middle of the area, but the 25-year-old skewed his shot wide of the mark.
With the final attack of the half, Eastleigh took the lead. Michael Kelly’s deep free-kick was headed back into the danger zone by Carter and Ousseynou Cisse tapped home from close-range.
Chesterfield came out firing in the second half as they looked for a route back into the game; Cooper found Mandeville with a teasing cross, but McDonnell made a fantastic save from the midfielder’s sliding attempt.
Mandeville would be the one to find the equaliser though as his low cross found the back of the net via a deflection.
The Spireites piled on the pressure after as they chased the lead. Jamie Grimes saw his header brilliantly saved by McDonnell and Ollie Banks headed wide of the mark from Jeff King’s free-kick.
Despite the Spireites being on top, it was the home side that would once again strike late into a half.
Ryan Hill’s deep corner was headed into the back of the net by Camp despite Akinola’s best attempts to clear the ball away.
Chesterfield: Fitzsimons, King, Cook (Williams 62), Grimes ©, Horton, Akinola, Banks, Mandeville, Asante (Uchegbulam 69), Cooper (Quigley 72), Tshimanga.
Unused Substitutes: Clements, Jones.