The visitors scored the only goal of the game, eight minutes into the second half through Martin Gritton.
The forward picked up a delightfully weighted pass from strike partner Jack Lester and didn't need to break stride as he curled the ball past Ben Hamer and into the far corner.
The home side should have been ahead at that point as they missed a number of good chances in the first period and were made to rue them.
Top scorer Charlie MacDonald missed the best of them when his header from eight yards out was weak and saved low by Chesterfield goalkeeper Tommy Lee.
Jordan Rhodes failed to convert a low MacDonald cross when only six yards out and also fired over from close range.
Even after Chesterfield's goal Brentford had chances. Lee tipped an acrobatic Marcus Bean effort over the top and MacDonald shot over from close range after good work by Ryan Dickson.
In the end Chesterfield could have won more comfortably with Drew Talbot missing a number of chances.
The worst of them was when he picked up a weak John Halls back-pass and rounded Hamer only to hit the post with the goal gaping. He also forced Hamer into a low save and curled an effort narrowly wide.
Gritton headed wide when well-placed for the visitors and Lee hit a Dickson cross-cum-shot around the post when it looked to be arrowing in.
Substitute Sam Williams turned an effort wide from close range as Brentford pushed for an equaliser and David Hunt screwed a shot wide from ten yards.
Chesterfield's defence held firm and they repelled everything Brentford could throw at them to seal a shock away victory and keep their slim play-off hopes alive.