About 100 people, including more than a dozen in school uniforms, marched from the State Library and briefly ran across some of city's busiest intersections as part of the School Strike for Palestine.
Trams were stopped along Swanston, Bourke and Collins St as the group made their way through the CBD on Tuesday afternoon.
Protesters ran up the steps of Flinders Street station, with many seen jumping over ticket barriers.
Earlier, year 12 student and protest organiser Giselle Nayef said their "global movement" had put pressure on governments, pointing to a decision by the Victorian state government not to renew a defence memorandum of understanding with Israel.
"So long as there is oppression caused by our ruling classes, we belong here on the streets, not in school, not anywhere else, and that's why we're gonna keep showing up," Ms Nayef told reporters.
"This is the biggest crime of our generation, and we will not just sit and ignore it and listen to when our teachers tell us it's too complicated, listen to when our politicians tell us they've got it under control."
Student Gisele Nayef joined hundreds of protesters at the School Strike for Palestine. (Joel Carrett/AAP PHOTOS)
Rally speakers included National Students for Palestine convenor Jasmine Duff and former Victorian Greens leader Samantha Ratnam, the party's candidate for the federal seat of Wills.
Year 11 pupil Maja said there is a lot of passion for the cause among students and she left school two hours early to attend the protest with the support of her mother, Maggie.
"Mum is really supportive about it, I saw (flyers) taped at my school everywhere and it's for a good cause," she told AAP.
"It's just two hours, let's just leave school, I can always catch up."
Maggie attended with her daughter to ensure there was no "trouble" while eyeing off the visible police presence.
"I encourage both of my daughters to be powerful and strong as women and as supporters of others," she said.
"Children are getting killed every day and it's a very sad state of events."
Protesters swarmed Flinders Street Station during the School Strike for Palestine. (Joel Carrett/AAP PHOTOS)
It comes a day after a man was arrested when tensions ran high outside a Melbourne synagogue, as pro-Israel supporters gathered to counter a pro-Palestine crowd.
Victoria Police said 150 people gathered at Caulfield Shule on Monday evening after a planned pro-Palestine protest was called off at the 11th hour due safety concerns.
A 39-year-old Brunswick East man is expected to be charged with breaching the peace and failing to move on.
He was bailed to appear at Moorabbin Magistrates Court on March 14.
There was a strong police presence near the synagogue in Caulfield North, in Melbourne's southeast, where a sea of Israeli flags far outnumbered those for Palestine.
Specialist police encircled a small group of pro-Palestine supporters, who held anti-Zionist signs, in an attempt to keep them from the larger pro-Israel group.
The planned demonstration was in response to a talk organised by the Australia/Israel and Jewish Affairs Council that included former Israeli justice minister Ayelet Shaked, whose visa into Australia was reportedly rejected.
Victoria's Premier Jacinta Allan said there is "no place for protests of this kind anywhere in the state of Victoria" as she unveiled anti-vilification and social cohesion reforms on Tuesday.
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