The train, pulled by a tractor made to look like a tank engine, first ran from Stirling Castle, through the cobbled streets of Top Of The Town area, down to the city centre and back, last summer.
Businesses, past whose premises visitors to the Castle, the ancient seat of the Stuart kings, normally had to walk, claim the train dramatically reduced footfall and therefore takings.
Nearly 70 businesses have signed a petition calling for the end of the seasonal service.
The petition criticises Go Forth Stirling, the city’s Business Improvement District body, for its handling of their complaints and wants the impending return of the land train to be put on hold.
The train was introduced last year by Go Forth Stirling in the hope it would encourage tourists to visit different parts of the city.
Over Christmas, driven by a man dressed up as Santa Claus, the service ran from near the Thistles Shopping Centre in Murray Place to the Castle Esplanade, before returning to Murray Place, and was free.
Businesses have been told that when it is re-introduced in the summer there will be a £2 flat fare.
Alan Waldron, owner of Stirling Bagpipes on historic Broad Street, near the castle, and organiser of the petition, said businesses were not consulted about the train’s introduction.
Mr Waldron, 48, said: “We’ve been round 70 shops so far, and only two have said that they like it. It’s totally kitsch and tasteless.
“Tourists come out of the Reformation castle and get on this Mickey Mouse train which whisks them straight away from the shops and businesses near the castle and deposits them near Debenhams.
“Businesses near the castle have totally lost out on ‘strollers’ since this train was introduced.
“I can see why the manager of Debenhams likes it, but it’s no good for anyone else.”
Mr Waldron now hopes local Conservative MP Stephen Kerr will back his campaign against the train.
Jane Hamilton, owner of Broad Street lifestyle and antiques store Sage, said: “I don’t actually think the idea for a land train is that bad but the BID has been completely inflexible with us.
“The location is great for the big shops as it brings customers right to them. So it works for them, but it has had a profound effect on small businesses.”
Go Forth BID director John Walton said he was “disappointed” to hear that traders were unhappy.