A flight crew checking the cabin of a Qantas plane before take-off found rats in a compartment holding medical equipment, grounding the plane for more than a day, a spokeswoman said.
Crews did a visual check of the plane on Tuesday afternoon and found no more rats or any damage.
The rodents had been in a cabinet holding a defibrillator. The plane returned to service on Thursday morning.
Passengers had not yet boarded the Sydney-to-Brisbane flight and were instead put on another plane.
A Qantas spokeswoman called the incident "very unusual".
But Scott Connolly of the Transportation Workers Union told Australian Broadcasting Corp radio that members have had concerns about hygiene and sanitation on Qantas flights.
The spokeswoman denied that was the case. "This is a very irregular occurrence," she said.
Qantas, Australia's national carrier, has experienced a series of troubles in recent months, including an engine explosion and forced landings.
Earlier this week, a Qantas flight from Dallas, Texas, to Australia made an unscheduled stop on a Pacific island after pilots feared they might run out of fuel because of strong headwinds.
The Qantas spokeswoman said the airline was committed to the route - which is new and one of the world's longest for Boeing 747 jets.