They are warning there will be thousands of job losses unless the First Minister amends the situation.
And they have written to Nicola Sturgeon calling for immediate changes.
The rules mean hotels can serve evening meals to residents but not with alcohol, although wedding guests at the same venue can continue to be served alcohol.
Among the 100 hoteliers who have signed the letter are David Ibbotson, of Knockinaam Lodge at Portpatrick, and Ian McAndrew, of Blackaddie Country House Hotel at Sanquhar.
It says: “Not being able to sell alcohol in public areas to hotel residents in Scotland negatively impacts their stay and future guests are already starting to cancel their bookings. This measure in particular is threatening the small thread of revenue – a lifeline for many – which still exists for hotel businesses in Scotland at this difficult time.
“We urge you to reconsider this and allow hotel guests, staying a minimum of one night, to consume alcohol in all settings, not simply room service alone. In addition, we believe that we should be able to serve non-residents until 6 pm, as a café is allowed to do.
“If there is no change, we have no doubt that we will suffer deeper losses. We are talking about trying to survive, not about profitability.”