The petition warns many guesthouses and B&Bs face financial ruin and calls for "fairness" in the support on offer.
Restaurants, bars, pubs, cafes and hotels are all eligible for the hospitality top-up payment, but B&Bs, guesthouses, self-catering accommodation, campsites and caravan parks are not.
The Scottish Guest House and B&B Alliance, set up in response to the withholding of the funding, said their sector was a core part of Scotland’s tourism and hospitality industry and supported 3,000 jobs in Scotland.
But they said denying them the grant was a fresh financial blow and claimed many otherwise viable operations faced job losses and permanent closure.
Alliance member Fraser Mathieson, who is based in Inverness, said: “This grant could potentially save thousands of jobs that are at risk within this group of businesses, thereby helping to save the wider tourism sector in Scotland, which depends on local accommodation to sustain it, and to sustain local economies.
"The travel restrictions brought in during October has seen trade drop to almost zero. Other local small businesses not nearly as badly affected as accommodation providers have received this lifeline top-up support and we have not.“With tourism being so important to the Scottish economy we cannot understand why we have been excluded especially when the other nations in the UK are supporting our sector so well.”
Karen McClelland from Ayrshire said her local group had initiated the petition because all other attempts to raise the issue with the Scottish Government had hit a brickwall.
"I believe that if they fully understood the issues and lack of equality with regard to this sector, they would review their decision and award us this grant, putting us on an equal footing with other businesses of a similar size, with similar overheads.”
Guesthouses in Edinburgh have also protested over the grant exclusion. Ross Birnie, who runs 23 Mayfield, said he feared lack of support could drive small businesses out of buiness, leaving larger operators in a dominant position.
The Scottish Government has said it has limited resources and therefore must target its action.