Last year, 10 MPs and senators were removed from office for violating a constitutional rule that prohibits federal political candidates from being dual nationals.
On Wednesday, Senator Katy Gallagher was ruled ineligible by a court in what was widely viewed as a fresh test case.
Four other politicians who were under scrutiny resigned after the verdict.
Ms Gallagher and lower house MPs Justine Keay, Josh Wilson and Susan Lamb are members of the opposition Labor party. The fifth MP, Rebekha Sharkie, is part of minor party Centre Alliance.
The dual citizenship saga has destabilised Australian politics since last July, at times threatening Prime Minister Malcolm Turnbull's majority in the House of Representatives.
Mr Turnbull could increase his majority if he wins any of the vacant lower house seats.
The High Court of Australia ruled that Ms Gallagher's seat would be filled by counting back votes from the 2016 federal election.
The other politicians are expected be replaced in the same way, or through a by-election.
Ms Gallagher had referred herself to the court last year after questions emerged over whether she had renounced her British citizenship in time to run for office.
"To the people of the [Australian Capital Territory], I'm very sorry that this disruption has occurred to one of your federal representatives," she said on Wednesday.
Labor leader Bill Shorten said three of the ousted MPs would seek re-election.
Mr Shorten said his party had relied on an older interpretation of Section 44(a), a much-debated rule in the constitution.
"The High Court has made the decision - these are the facts we've got to deal with," he said.
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