Leaders Brentford's title hopes took a knock as Martin Gritton's goal gave Chesterfield the points.
The Bees showed the effects of three games in a week and Charlie MacDonald wasted their best chance when he headed straight at keeper Tommy Lee.
Gritton's 53rd-minute winner came with a neatly curled finish from Jack Lester's intelligent pass.
Drew Talbot could have wrapped it up on the hour when he rounded Bees keeper Ben Hamer only to hit the upright.
Brentford: Hamer, Halls, Phillips, Bennett, Dickson, Newton (Marvin Williams 71), Hunt, Bean, Wood (Johnson 60), MacDonald, Rhodes (Sam Williams 51). Subs Not Used: Simon Brown, Osborne.
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Chesterfield:
Lee, Wilson, Hall, Downes, Robertson, Lowry, Montrose, Niven, Talbot, Gritton, Lester. Subs Not Used: Austin, Currie, Kerry, Gray, Till.
Goals: Gritton 53.
Att: 4,541
Ref: Mike Russell (Hertfordshire).
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