Organisers from the group Friends of Palestine WA vowed to disrupt the activities of a ZIM ship due to use the port on Saturday afternoon.
The group established blocks of two roads leading into the Port, but also complied with police orders to let trucks through to avoid committing a criminal offence.
Grace Brooks from Trade Unionists For Palestine said the company was being targeted because it had been used in the past to ship weapons.
Protesters are planning to remain at the Port until 9pm on Saturday.
"As trade unionists who care about peace, we cannot stand by as our fellow workers in Palestine are being subjected to what can only be described as genocide," Ms Brooks said.
"ZIM is complicit in this genocide and until that changes we will disrupt their business."
A large police presence is monitoring the protest activity, due to last 24 hours.
Organisers told AAP the ZIM ship's arrival had been delayed until 10.45pm and they would reassess if the time change would impact the length of the action.
It's the latest action taken by protesters who have been on the streets every weekend across the country since the attack by Hamas militants on Israel in early October sparked the latest wave of unrest in the Middle East.
Similar action taken by protesters targeting ZIM ships at the Port of Botany in NSW last month ended in 23 arrests.
Police blamed "professional protesters" for the violence which included scuffles with officers after about 400 people took part.
The Fremantle protest also comes after talks to extend a week-old truce between Israel and Hamas collapsed, prompting a resumption of hostilities.
Israeli warplanes pounded Gaza after the deadline lapsed shortly after dawn on Friday while sirens blared across southern Israel as militants fired rockets from the coastal enclave into towns.Â
Hamas said it had targeted Tel Aviv but there were no reports of casualties or damage there.
By the evening, Gaza health officials said Israeli air strikes had killed 184 people, wounded at least 589 others and hit more than 20 houses.
