The Spireites fell to their second league defeat of the season after a 3-2 loss to Maidenhead United at York Road.
The hosts took a 2-0 lead within the first 20 minutes before Jeff King halved the deficit just before the break.
Kabongo Tshimanga levelled the scores in the second half but Josh Kelly's second of the game gave the Blues a first league loss since September.
James Rowe made two changes to the side that battered Barnet on Saturday as Jim Kellermann and Alex Whittle replaced Akwasi Asante and Calvin Miller.
The hosts took the lead after seven minutes as Kelly curled an effort from 20-yards past Scott Loach.
Kellermann was presented with a golden chance a few minutes later; a poor back pass allowed the midfielder through on goal but Dan Gyollai used his frame to pull of a smart save.
By the 20th minute, Chesterfield found themselves two-goals down. Dan Sparkes powered his 20-yard free-kick past Loach and into the back of the net.
The rest of the first half didn't produce much in the way of chances until added time. King took aim and sent a piledriver of a free-kick into the top-left corner - his second in as many games.
The Spireites came out with a renewed sense of optimism. Asante came on for Kellermann during the break.
Just before the hour mark, Tshimanga popped up with the equaliser; Luke Croll found the striker before he prodded home from close-range for his 21st league goal of the season.
Chesterfield came very close to completing the comeback soon after. Tshimanga sent one effort just over the bar and a King corner somehow was failed to be headed home at the back post.
However, Maidenhead soon found their stride again and took the lead with just ten minutes to go. An in-swinging corner was flicked to the back post and Kelly was there to nod home his second of the evening.
Unfortunately, the Spireites failed to really threaten the home goal after and were condmened to just a second defeat in the National League.
Rowe's side also failed to return to the top of the league with the result but have a chance to put things right on Saturday with a home game against Aldershot Town.
Chesterfield (3-4-2-1): Loach; Williams, Grimes (Payne 82), Croll; King, Weston (C), Oyeleke, Whittle; Mandeville (Khan 73), Kellermann (Asante 46); Tshimanga.
Unused Substitutes: Miller, Maguire.
Attendance: 1,232 (322 away).