Earlier this week it was announced a deal was almost secure to keep "Spy Booth" in the town, after campaigners raised the money to buy it.
The piece, depicting men "snooping" on a phone box, appeared in April, three miles from government listening post GCHQ.
The local businessman, who has agreed to pay for the artwork, said he was "confident" it could be saved.
Jenny Lyon, whose boyfriend David Houghton spotted the vandalism on an early morning run, said: "There appears to be silver writing all over the three spies. It's completely ruined. It's awful.
"People have worked so hard to keep it here and then somebody overnight has ruined it for everybody."
Businessman Hekmat Kaveh, who agreed to pay an undisclosed amount to keep the artwork in place, said he believed it could be saved.
"I believe the original paint had some anti-graffiti products in it and also an anti-graffiti product was put over the top of it. I am confident that it will come off," he said.
Mr Kaveh said he was "rather surprised" local people had begun trying to remove the silver paint with a brush. "I would much rather get the professionals onto it," he said.
Experts will be called in to complete the clean-up work on Saturday and Perspex will be put over it for protection.
Gloucestershire Police said officers were carrying out inquiries and appealed for anyone who witnessed the act of vandalism to get in touch.
Campaigners have been trying to keep the artwork in situ on the corner of Fairview Road and Hewlett Road after owners of the house claimed it had been sold and workmen arrived to remove it.
Last month, Cheltenham Borough Council issued a temporary stop notice preventing further removal work from taking place on the Grade II* listed building.
A Banksy artwork painted on the wall of a house in Cheltenham has been defaced.