First Minister Humza Yousaf has ruled out a further delay to short-term let licensing.
All short-term let (STL) accommodation must have a licence and those who fail to apply can be fined up to £2,500.
The deadline has already been pushed back by six months to October 1, but some in the sector have asked for more time.
STL operators have warned the new rules could negatively affect the economy, tourism and jobs.
The Scottish Bed and Breakfast Association has urged the first minister to delay the scheme, arguing it will hit tourism and jobs.
It has warned that two-thirds of Scottish B&B owners are considering closing over the rules.
No fresh extension
But the first minister told the BBC: "There will not be another extension to the deadline.
"It is the right thing to do to bring this licensing scheme in. There has, of course, been an extension already."
Mr Yousaf added that he understood there were concerns about the scheme.
A recent report to an Edinburgh City Council committee suggested up to 80% of STL properties could be lost in the city.
"I know that there are some concerns," Mr Yousaf said.
"We continue to work with the sector, in fact cabinet secretary Neil Gray met with the sector, I think it was just last week. We'll continue to engage with the sector where we can but there'll be no more extensions."