Campaigners want Historic Scotland to call in the proposed revamp of the A-listed building after it was rushed through the city's planning committee without any debate last week.
City council officials insist they intend to start work next month to ensure the new-look building is ready for the 2012 Cultural Olympiad, which is being staged in the run-up to that year's Festival Fringe.
But promoters who have staged Fringe shows in the building for the last 30 years are demanding the plans are sent back to the drawing board, over fears about the impact on the venue of proposed shops and a fine-dining restaurant.
The council, which is expected to give a final decision on the project on Thursday, insists a full-scale refurbishment of the venue is long overdue and that the changes will help bring the building into more use 52 weeks of the year.
But Assembly Theatre claims five spaces used to house Fringe shows will be lost forever if the current plans go ahead.
Supporters have mounted a campaign to lobby Historic Scotland, which must approve major changes to a listed building, to call in the plans and pass them to the Scottish Government for a final decision.
Assembly Theatre has also written to thousands of people on its ticket database, urging them to lobby councillors on its behalf.
The council insists it does not plan to refer its proposed scheme to the government, even though it has a heavy financial interest in the refurbishment going ahead, despite more than 6,600 people backing an online campaign against the development.
But the council is legally obliged to seek official approval from Historic Scotland, which pointed out that it had not yet seen the latest plans for the Assembly Rooms, insisting that it had agreed only "in principle" to support the restoration.
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Last night William Burdett-Coutts, artistic director of Assembly Theatre, said he would be writing to both Historic Scotland and the Scottish Government to try to have the council's scheme subjected to further scrutiny.
He told The Scotsman: "The Assembly Rooms is one of the most iconic buildings in Edinburgh and it is clear the council has not taken account of public opinion.
"I don't understand the rush to get these plans through and I know there are people within the council who are very concerned about the way that this has been handled."
A spokeswoman for Historic Scotland, which has admitted having "very strong concerns" about some elements of the council's plans, said: "We understand revisions have been made, although until the city council formally consults us on the finalised plans we can not comment further."A spokeswoman for the council said there were no plans to contact the Scottish Government about its proposals, because they did not represent a "significant departure from our development plan".