The Football Association deemed Georg Margreitter was not registered to feature in Saturday's 1-0 win over MK Dons, despite having been registered for league action after signing temporarily at the Proact Stadium from Wolves.
Chesterfield remained in the draw for the third round, and were paired with the winner of Scunthorpe United and Worcester City's second-round replay.
However, their participation in that fixture has been thrown into doubt after the FA revealed a charge had been brought to Paul Cook's side.
A statement read: "The FA has charged Chesterfield FC with a breach of FA Cup rules, after allegedly fielding an ineligible player in their FA Cup second round proper fixture against Milton Keynes Dons.
"MK Dons were beaten 1-0 at home by Chesterfield on Saturday 6 December 2014, during which fixture Georg Margreitter featured for the away side.
"It is alleged that Margreitter - on loan to Chesterfield from Wolverhampton Wanderers - played in the fixture without the necessary written permission having been received by The FA as at the 1200BST deadline on Friday 5 December 2014.
"As a consequence it is alleged they are in breach of FA Cup Rule 15(j)(i) for fielding an ineligible player.
"Chesterfield FC has until 1700BST on Thursday 11 December to reply to the charge."
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