Rex Airlines is to make vaccination compulsory for staff who deal with customers. It's given them until November 1 to comply.
Qantas has already indicated that it will do the same.
The staff covered would be those at check-ins, and all pilots and cabin crew.
The company runs direct flights from Canberra to both Sydney and Melbourne, with connections to a raft of smaller regional airports.
"As we provide an essential service operating to regional centres and remote communities throughout Australia, it is incumbent upon us to do whatever we can to help those residents remain safe and healthy," the company's deputy chairman, John Sharp, said.
"We have a duty of care to both our passengers and staff to provide the safest possible environment."
The company surveyed staff and said that the overwhelming majority supported vaccination.
It will not insist on office staff having the jab, however. Unvaccinated "frontline" staff would be offered roles away from the public. "Unvaccinated office staff will be required to wear a mask while at work," Mr Sharp said.
A month ago, Qantas chief executive Alan Joyce said his staff would also have to be vaccinated.
"Having a fully vaccinated workforce will safeguard our people against the virus but also protect our customers and the communities we fly to," Mr Joyce said.
All Qantas's "frontline" workers would have to be vaccinated by November 15 and the rest by the end of March next year.
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At Canberra Airport itself, there is also a vaccination mandate.
The airport's boss Stephen Byron wants to be able to assure state premiers that people who travel through the airport do not present a significant risk of taking the virus with them.
The vaccination rule would also apply to contractors like cleaning companies and baggage handlers.
"The staff are absolutely on board and we're seeing it's not just for our own staff," My Byron said.