Division One leaders Belper Meadows beat them by 16 runs at Queen's Park.
This was Chesterfield's third defeat in succession and it means they drop from the crucial second promotion spot to fourth.
Belper won the toss on a pitch that provided a good balance between bat and ball.
With rain threatening - showers did cause two short stoppages - the usual thinking is to field first.
But Belper took the bold decision to bat - and justified it with positive stroke-play.
Openers Sahd Shafiq and Jamie Rowland both drove well as they took 10 off the first over and 52 off the first eight. That gave Belper a firm grip on the match and Chesterfield were never really able to loosen it.
Reece Johnson and Muhammad Zaroob slowed the scoring rate but Chris Sheldon's 54 made sure the visitors did not waste their good start.
The only time Chesterfield looked like getting back on level terms was when spinners Sam Fawcett and Ben Slater bowled in tandem.
Slow left-armer Fawcett took 3-29, including having David Whiteley smartly stumped by out-of-retirement wicket-keeper Steve McCarroll - and off-spinner Slater 3-30. But Belper's 200-9 off 50 overs was a challenging target.
Chesterfield began brightly and had reached 35 in the ninth over when Slater (22) pulled a short ball and was caught on the leg side.
Against good bowling and well-set fields, the run rate dried up. Off-spinner Chris Stevenson (2-29) bowled beautifully, his first four overs costing just one single.
Zaroob, with the help of some inventive shots, made a fluent 24 before Stevenson struck a decisive double blow.
Zaroob holed out at long-off from a tantalisingly-flighted delivery and, in his next over, Stevenson beat Luke Westwell's forward defence and bowled him for 25.
Thereafter the run rate required rose steadily and, although eight Chesterfield batsmen reached double figures, nobody went on to play a substantial innings.
Mitch Adlington hit the final ball for a straight four but Chesterfield were restricted to 184-7.
With three matches to play and 22 points for a win, Chesterfield - on 272 - are 13 behind second-placed Sawley.
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