Djab Wurrung elder Marjorie Thorpe is challenging the Western Highway duplication near Ararat in the state's southwest, seeking a further injunction over the removal of trees of cultural heritage.

She argues that as a Djab Wurrung descendent and elder it's her responsibility as a custodian of the land to protect trees and areas of cultural significance.

Six trees have been earmarked for protection in the challenge, which is being fought in Victoria's Supreme Court.

Barrister Ron Merkel QC is representing Ms Thorpe and the community and says multiple reports by archaeologists and anthropologists show the trees are significant Aboriginal places.

He said the case came down to a custodian seeking to protect the heritage of her people versus sacrificing the heritage for a quicker and safer highway duplication project.

At issue is 12.5km of the Western Highway - the main route between Melbourne and Adelaide. Other sections have already been completed.

While six trees of significance have been identified, authorities say only one is actually earmarked for removal and not before a Federal Court trial on the overall case.

Mr Merkel said the tree was on the road under the current proposal and would have to be removed.

Road authorities have considered works that protect the roots, and therefore the trees won't be harmed, he said.

But his clients consider the area to be of significant Aboriginal cultural and heritage.

"The land belongs to the people but the people belong to the land," he said.

"This case is a really important test of how Aboriginal heritage is protected in Victoria."

The Aboriginal Heritage Act makes it an offence for someone to harm Aboriginal cultural heritage unless acting in accordance with one of a series of exemptions, including a permit or approved management plan, or land management agreement.

He said on no view of the existing plan was there a warrant to destroy cultural heritage.

But Richard Attiwill QC for the state and project authorities criticised Mr Merkel for not mentioning the overall case's prospects for success - which he says are next to zero.

Works have already commenced, land has been acquired and money spent, he said. Ms Thorpe had allowed those works to happen, signing an agreement in October last year.

The application is also for a broad injunction that would permanently prevent all works in a particular area.

"That is an indefinite injunction that will forever restrain the upgrade of the Western Highway," he said.

"The prospect of a court granting that relief is, we say, so low as to be a completely insufficient prospect - it just won't happen."

The hearing is due to continue on Friday.

Australian Associated Press