Police have issued 17 move-on notices to protesters opposed to the Perth Freight Link and officers will remain at the site while work is undertaken.
About 60 protesters opposed to the controversial Perth Freight Link have reluctantly left the site after police issued 17 move-on notices, while Premier Colin Barnett insists the environment will be improved by the road.
The first stage of the project, the planned Roe 8 highway extension, will cut through the Beeliar Wetlands, angering activists who claim it will destroy the area, which is a habitat for the endangered Carnaby's black cockatoo.
Protesters are also concerned about the impact on rainbow bee-eaters, protected migratory birds that use the wetlands to nest until the end of February.
Fencing was put up at the site on Monday with protesters and officers watching on, but part of the fence was knocked down on Tuesday.
Rethink the Link campaign manager Kim Dravnieks said protesters arrived to find the fence blown down by wind, and sat on the fencing, prompting the move-on notices.
Those involved will have to stay away from the site for 24 hours and a police spokeswoman said officers would remain there while work was being undertaken.
Ms Dravnieks said protesters would also be at the site every day, particularly to keep the community informed on work being done as there had been little consultation.
"Different people have been told different things," she told AAP.
Ms Dravnieks had been told trees would be taken down, whereas others had been told fencing was put up so animals could be trapped and relocated.
The Conservation Council of WA encouraged the public via a video on social media to call the premier and transport minister Bill Marmion, and ask for all work to stop.
Director Piers Verstegen said he had been told the premier's office was inundated with calls.
Mr Barnett insisted the highway would not damage the wetlands environment and about $45 million had been put towards designing the road to ensure it was protected.
"If anything, the actual water quality, the environment and the habitat will actually be improved," he told 6PR radio.
"This has been delayed by legal actions and the like - they are basically exhausted as far as I can see - and construction will go ahead."
Save Beeliar Wetlands activists will head to the High Court on December 16 for a special leave hearing, where it will be decided if a full hearing will proceed for their legal challenge against the project.
Meanwhile, the group says the federal department of the environment is now investigating the absence of the rainbow bee-eater from the Roe 8 fauna management plan.
Save Beeliar Wetlands has mapped 29 active borrows, and say contractors are driving, walking and erecting fencing in those areas.