South Ayrshire Council has written to 47 householders advising them to settle their arrears or risk court proceedings which could lead to them being evicted from their homes.
The letter also told them they faced £400 in legal costs and that if they were parents, "children's services would be informed".
Anti-bedroom tax campaigners criticised the council's actions.
"This is horrendous harassment of tenants over unpaid rent of just a few hundred pounds in many cases," said Matt Dobson, a member of the Scottish Anti-Bedroom Tax Federation. "People are terrified of being evicted."
South Ayrshire is the latest local authority to write to tenants over unpaid rent connected to the bedroom tax, a controversial welfare change brought in by the Coalition Government.
Under the policy, council or housing association tenants who are deemed to have an extra bedroom have their housing benefit cut by 14%.
In August, North Lanarkshire Council backed down from evicting a disabled tenant after a public meeting attended by 100 people. Councillor Jim McCabe, the leader of the Labour-led council, pledged not to evict any tenant in arrears because of the bedroom tax as long as they engaged with the council and the arrears were solely due to the tax.
Last night a spokeswoman for South Ayrshire Council said 1200 tenants were affected by the under-occupancy rule, with letters being sent to 47 who had "failed to engage" with the council over the unpaid rent.
"Rent is a payment that is lawfully due from tenants and this income is required to ensure we can continue to provide frontline housing services and also carry out planned investment and improvement works to our housing stock," she said.
"We have a very positive and productive relationship with most of our tenants, are happy to discuss any issues they may have, and are committed to supporting those who can't manage their rent payments as a result of the benefit changes.
"However, we need to have a discussion with the individual to achieve this. And when people refuse to respond to our attempts to contact them, won't engage with us, and won't pay, they leave us with no choice but to escalate matters in line with our council-approved policies."