Town had been due to take on the National League North side at the Technique Stadium this Saturday.
As a result of Chesterfield’s withdrawal, Guiseley will progress to the fourth round.
The Blues are set to play Notts County in the National League on Tuesday night. No announcement has been made about that fixture yet.
Chesterfield withdrew from last season’s competition for the same reason.
On Wednesday the UK hit a record for daily coronavirus infections since the pandemic began.
Many other matches across the country have been postponed.
"A Covid event has resulted in Saturday’s Buildbase FA Trophy tie at home to Guiseley being called-off,” the Spireites said in a statement.
“The Spireites have now taken the decision to withdraw from the competition.
“An announcement regarding refunds for tickets purchased for the fixture will be made soon.”