The court ruled that the two dozen settlers had until 11am (0800 GMT) on Friday to leave the three-storey building, whose ownership is claimed by Palestinians.
The West Bank settlers argued they had purchased the property legally from the owners.
"We advise those in the house to leave of their own volition in order to avoid needless confrontation," the three-justice panel said in its ruling.
Hebron, a frequent flashpoint of Israeli-Palestinian violence, is home to 130,000 Palestinians and 400 Jews.
The city is holy to Muslims and Jews as the burial place of biblical patriarchs.
The court did not decide who owned the house, ordering it to be boarded up until that dispute was resolved.
David Wilder, a spokesman for the settlers, said there had not been a decision on whether to honour the court's eviction notice.
"We will sit and make a decision later this evening on what we plan on doing," he said.
A settler chalks the words
'Shapira's house' across a sill
The settlers moved into the house on April 6.
The dispute arose three months after Israel forced nine settler families to leave an unauthorised enclave they had set up in Hebron's former wholesale marketplace.
Those settlers had agreed to leave peacefully after a standoff that lasted several weeks.
At the time, they said they were leaving on the understanding that the Israeli authorities would allow them to expand the Hebron enclave at a later date.
The dispute is an example of the challenges which Ehud Olmert, the Israeli prime minister, will face over his plan to set Israel's borders by 2010.
As part of the plan, Olmert has pledged to evacuate isolated Jewish settlements in the West Bank while building up several larger blocs in the occupied territory, a move Palestinians said would annex land and deny them a viable state.
The World Court has branded as illegal Israeli settlements built on land captured in the 1967 Middle East war. Israel disputes this.