The club has been without a permanent base since its Meadow Park home was ruined by flooding in 2007.
A ground share was agreed with Cheltenham Town FC (CTFC) in 2010 but the Tigers now owe the money for unpaid rent and other expenses.
CTFC chairman Paul Baker said the debt cannot be written off and has asked for the money by the end of March.
He said: "We have got an ultimatum that if they don't clear their debt by the end of March and satisfy us that they can meet the ongoing rental payments that are due then we won't be able to let them continue using our pitch and we won't be prepared to enter into an agreement for them next season.
"We will give them as much help and assistance as we can, but the bottom line is that we have a business to run here. We were banking on receiving that income."
The chairman of Gloucester City AFC, Nigel Hughes, said the club was "desperately" trying to generate some investment.
"If we had the money we'd have paid," he said.
"Paul Baker is quite right - it's a business that they are running at Cheltenham Town and if the shoe was on the other foot, as they say, I'm sure we'd have taken the same stance."
Mr Hughes added that the Blue Square North club was struggling to make any money at all on midweek home fixtures as only a few hundred supporters were attending the games.
Gloucester City AFC has been told it needs to settle a Â£20,000 debt by the end of the month or face eviction.