The group marched past the National Gallery of Victoria and the ABC’s Melbourne headquarters, blocking roads and local traffic.
Initially greeted with a minimal police presence, officers and mounted police began to move in close to the group just after 1:30pm, breaking them up into smaller groups, before they began to make arrests.
By 2pm, much of the anti-lockdown crowd had dispersed among the parklands.
The gathering follows the announcement yesterday that from October 15, Victorians working in occupations on the authorised workers' list must have had at least vaccine dose - unless they have an exemption.
The list of occupations include emergency services, personal trainers, marriage celebrants, journalists and media workers, retail workers operating click and collect services and members of the public service undertaking essential work, including politicians.
The protest coincides with Victoria’s highest day of case numbers, blamed in part on illegal social gatherings from last weekend.
In the past 24 hours, 1,488 cases were recorded and two deaths.
Victoria sets new record with 1,488 local COVID-19 cases and two deaths
When asked about today’s protest by reporters, COVID-19 commander Jeroen Weimar expressed his frustration at their actions.
“Why? Really, I mean, we get it. We get the fact that people are frustrated, we get the fact that we're upset. You're not helping yourself, you're not helping anybody,” he said.
“At best, you run the risk of generating more transmission. At worst, you know you're creating, you're fuelling a bonfire, that's already on fire. I just think it's a really unhelpful thing to do.”
Today’s protests appear the largest in size since protesters occupied the Shrine of Remembrance for hours on Wednesday 22 September.
That gathering was broken up by Victoria Police with tear gas and rubber bullets.