Around 250 locals of Little River, near Werribee, gathered in protest at a proposed Pacific National freight terminal that would handle as many as two million containers a year.
The hub, which has not yet been approved for a 550-hectare site, would cost between $3 billion and $5 billion and take 25 years to build.
Little River, with a population of 1400, has formed an action group and gathered 5000 signatures so far opposing the terminal.
Grassland ecologist Dr Megan O'Shea said the site had been planned in an environmentally sensitive area.
"A massive freight hub will sever the link between Western Grassland Reserve and the internationally recognised Ramsar wetlands of Port Phillip Bay, impacting on the threatened plants and animals," Dr O'Shea said.
Earlier this year, federal environment minister Tanya Plibersek rejected Victorian state government plans to dredge the Western Port Ramsar wetlands to build a wind turbine assembly plant at the Port of Hastings.
Locals have photographed the endangered growling grass frog in Little River, while departmental surveys have found critically endangered golden sun moths on neighbouring land.
Other threatened animals such as the striped legless lizard and fat-tailed dunnarts have also reportedly been spotted nearby.
Grassy Plains Network facilitator Adrian Marshall said the freight terminal would impact the equivalent of around 75 football fields of rare volcanic plain grassland.
"99 per cent of Victoria's native grasslands have been destroyed. Why remove more?" Dr Marshall said.
"There has not been a proper environmental assessment of the site. What is the logic of putting a freight terminal there?"
Little River Action Group president Adrian Hamilton said if the terminal was built, 1500 trucks would pass through the area every day.
"This freight terminal will bring significant noise, light pollution and fumes emissions to our door," Mr Hamilton said.
"It makes no sense that residential development is banned on the green wedge, but they are proposing an industrial freight monolith in the centre of a fragile and threatened ecosystem."
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