Assistant manager Marc Richards has also left the club.
The Tigers sit 16th in the Vanarama National League South and have won just twice in their last 11 league games.
Player-coach Will Morford will be in charge for Saturday's home match against Eastbourne Borough.
Harris began his second spell as manager at the club in 2014, having previously been in charge from 2006-2008.
The club has been without its own ground for ten years after severe flooding forced them to move out. Work has began on rebuilding their stadium although and the club issued an update yesterday informing supporters that the next stage is to remove a telecommunications mast which is currently blocking any more progress.
Upon his departure, Harris told the club's website: “It’s with a heavy heart, that this morning I have resigned from Gloucester City Football Club, for those that know me it’s a decision I’ve not taken lightly, and it’s been something I have wrestled with for at least the last six months.
"I came back nearly four years ago, I have worked tirelessly with many good volunteers that shared my passion for the club, we’ve tried to hold the club together whilst maintaining our league status in a very professional league.
"At the end of last season, I expressed my concern publicly that the club could not continue without changing the structure and it shouldn’t keep relying on the goodwill of people that put in the endless hours a week that it takes to run a club at this level.
"Recently I surpassed 300 games managing this club, a record that I am proud of though there is no doubt the club needs to change.
"To say we have been punching above our weight is a massive understatement and the club needs some investment, some structure and business acumen if it is to maintain any hope of returning home at this level.
"We have lost a generation of supporters it’s nearly 11 years since we played in our City and many fans have become disillusioned during this time. I do feel for those supporters who travel away and away for home games I sincerely hope they understand my position.
"I informed the players after training on Tuesday evening of my decision and I have said to them they must go out and show a high standard, nothing more would please me than to see them win the game against Eastbourne Borough this weekend.
"I am indebted to Will Morford to committing to stay on in the interim to keep the players together until something is decided.
"Finally, I would like thank everyone that has supported myself and the club and seriously hope things will change for the better."
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