Martin Thacker and Jane Cooper, who joined the board when the community trust first took over the club in 2020, have now left.
The decision comes following the investment of Chesterfield brothers Phil and Ashley Kirk in March.
The pair will remain trustees of Chesterfield FC Community Trust.
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Thacker will continue to serve on the board of the National League.
The club said in a statement: “Their resignations came as a result of the loan by Phillip Kirk, in which community trust representation on the board is to be reduced. As part of this, Ashley Kirk joined the board and John Croot and Andy Fantom also became directors.”
Chairman Mike Goodwin said: “I would like to thank both Jane and Martin for their invaluable work during the takeover and in establishing us as a community club.
“I know both still have a lot to offer in the future with our wider work.”