Doc biodiversity ranger Jim Fyfe said motorcycle tracks had been seen twice recently at Hoopers Inlet, in Otago Peninsula.
The area was considered ecologically important in general, but had now also emerged as an important habitat for young pups of the threatened species, Mr Fyfe said.
Doc had conveyed its concerns to police and both agencies were keeping an eye on the area, he said.
"We’re certainly taking this pretty seriously," Mr Fyfe said.
"It hasn’t been something that’s been a problem for the last few years and we certainly don’t want to see it to become a problem now.
"If people do persist, they are very likely to get caught."
New Zealand sea lions have only recently returned to breed along the mainland’s southern coast, and Dunedin’s sea lion population produced a record 20 pups this breeding season.
Mr Fyfe said to find off-road tyre tracks in the area the pups were using, just as Doc was asking motorists in the area to take extra care and look out for the rare animals, was "very concerning".
Senior Constable Aaron Smith, of Portobello, said police were looking to speak with anyone who had information about the motorcycle tracks in the intertidal area.
Not only was the behaviour a safety risk for the threatened species, it also contravened Dunedin City Council bylaws, he said.
Driving or parking on Dunedin beaches was not permitted, except to launch or retrieve a boat, or in emergency situations, he said.
"Joy riding a motorcycle so close to the sea lion nursery is extremely disappointing and irresponsible," Snr Const Smith said.
Anyone found responsible could be prosecuted under the Reserves Act and fined up to $5000.
They could further be in breach of the Marine Mammals Protection Act, he said.
