Flitcroft was appointed as the permanent successor to Keith Hill in January and managed to save Barnsley from relegation to League One last season.
However, the Oakwell outfit have won just two games this term and Saturday's reverse against Birmingham City left the club bottom of the table and six points adrift of safety.
A statement on Barnsley's official website read: "Barnsley Football Club can confirm first team manager David Flitcroft has been relieved of his position.
"Technical coach Martin Scott and strength and conditioning coach Guy Proctor have also parted company with the club.
"Assistant Manager Micky Mellon has been placed in caretaker charge of first-team affairs, assisted by development coach Paul Heckingbottom in the meantime.
"There will be no further statements from the club until Wednesday 4th December 2013.
"The club would like to place on record their appreciation and thanks for what David, Martin and Guy have achieved."
Barnsley face back-to-back away games against Brighton and Hove Albion and Burnley next week before a home match against Yeovil Town, another relegation-threatened club.
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