Around 250 vehicles have been parked around the National Library, with police telling the anti-vaccine mandate protesters on Wednesday they were camping illegally.
Audio warnings were then given on Thursday before the move-on order was issued on Friday.
Most cars are leaving the area voluntarily but at least one has been towed.
A protester using a megaphone said the crowd would stand their ground but urged those who remained on foot to be peaceful.
ACT chief police officer Neil Gaughan says people are allowed to remain in the area and protest all they like, but cannot camp illegally.
"This is disruptive to Canberrans. That site was going to be used for events over the weekend and an event tonight," he told the ABC.
"The National Capital Authority has had to cancel that because of the fact that these people are illegally camping, so we are moving them on."
Items not moved may be seized by police.
The anti-vaccination mandate campaign, known as the Convoy to Canberra, has included a march on Parliament House on Monday and a rally outside the National Press Club on Tuesday where Prime Minister Scott Morrison was speaking.
Police used capsicum spray and made three arrests on Wednesday at the camp.
An ACT policing spokesman said one woman assaulted an officer and was arrested and charged.
Two men also tried to interfere with the woman's arrest and were also arrested.
Both men were charged with obstructing police.
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