The Vanarama National League North Tigers have announced the former Premier League winner and assistant manager Yan Klukowski have left the club this morning.
Flowers took over from Steve King this summer, stepping in after the club made the play-offs.
But the team sit second from bottom and on Saturday were humbled 3-0 at home to AFC Totton in the FA Cup.
City have already begun their search for Flowers’ replacement as they look to turn their fortunes around after a difficult first six weeks which has seen just one win.
The club said in a statement: “Gloucester City AFC can confirm that Tim Flowers has left his role as Manager by mutual agreement.
“To give a new manager a fresh start, his Assistant Manager Yan Klukowski has also left the club with immediate effect.
“The club would like to thank them for their efforts and will now focus on securing the right replacement to improve our fortunes on the pitch.”