More than 30 people were arrested after hundreds of protesters pushed down fencing to prevent clearing work from continuing for the controversial Perth Freight Link, which the premier says is "essential" for traffic.
Two people were arrested for chaining themselves to machinery on Thursday morning, as a woman protested in a tree to prevent bulldozing.
In total, police said they arrested 31 people and issued five move on notices.
The 39 charges include trespassing and obstructing public officers.
The $1.9 billion Liberal National government project was given the legal all clear on Monday when the Federal Court rejected the Save Beeliar Wetlands group's application for an injunction.
Premier Colin Barnett said the highway extension was "essential for efficient transport", would reduce traffic congestion and save lives.
"People have a democratic right to protest," he told reporters.
"They do not have a right, however, to break down fences and force themselves onto the site and interfere with people lawfully going about their business."
He said protest groups had cost the state a lot of money in police and court proceedings to delay construction.
Roe 8 and the broader Perth Freight Link road project is a major election issue with WA Labor vowing last week to cancel the contracts if elected.
Leader Mark McGowan said the government should not be building the road, and construction should be stopped until the public could have their say at the state election.
"It is economically, financially and environmentally irresponsible to continue with the construction of this road," he said.
Mr Barnett described Labor's plan as reckless and said it would send the wrong message that the state did not honour contracts.
City of Melville councillor Tim Barling and City of Fremantle councillor Sam Wainwright were among those arrested on Wednesday, with protesters wanting work to stop until the March state election.
State Environment Minister Albert Jacob said on Wednesday he was "comfortable" with land being cleared despite the fact clearing would be wasted if Labor wins office, rejecting calls to stop clearing until the state election.
Greens MP Lynn MacClaren said Mr Jacob should stand down.
Traditional owners have taken up residence at the Beeliar Wetlands protectors' camp, with Beeliar Aboriginal custodian Corina Abraham saying that by "desecrating this place, it's like the Barnett government is throwing sand in the face of our ancestors".
Community member Rachel Pemberton said Labor's position had given protesters hope, and they would continue to fight.
