Nearly 200 protesters gathered at the city's Civic Green on Saturday before marching down Liebig Street, chanting "sack Dan Andrews", 'kill the bill" and "save our children".
The largely peaceful protest - part of the World Wide Rally for Freedom against mandatory vaccination - began just after noon and required just a small police presence.
Attendees carried signs saying 'your body is no longer your own' and 'if you need a pass to prove you are free then you are not'.
Both the Red Ensign and Eureka flag featured in the march, the use of which had been widely condemned by both the Returned and Services League of Australia and descendants of Eureka rebels.
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The rally comes as Victoria entered phase D of the National Plan on Friday, which saw the removal of density caps and mask-wearing in most settings.
Under the plan, non-vaccinated residents would not be allowed entry into non-essential retail or hospitality venues and only allowed take-away.
Health authorities expect the 90 per cent double-dose vaccination mark to be reached over the weekend.
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