Ethicist Leslie Cannold was the party's number two candidate for the Senate behind Assange. She would have likely taken his place in the upper house in the event the party was successful in the September 7 polls and its leader unable to return to Australia.
"Even if I stop campaigning this minute, remaining in my role implicitly invites voters to trust The Wikileaks Party," Cannold said in a statement.
"By staying in this role I am implicitly vouching for the worthiness of this party to receive the votes of the Australian people. I can no longer do this because I no longer believe it is true, and so I must resign."
Cannold said the way preferences were to be directed in New South Wales and Western Australia "exposed problems with the capacity of the party to sustain its democratic processes".
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She said others would also resign the party. After her announcement, Daniel Mathews, a member of the party's 11-person National Council -- which includes Assange and his father John Shipton -- also quit.
"I am afraid that my experiences with this party are not all positive," he said.
The WikiLeaks Party had vowed to be a party of transparency and justice and one which would scrutinise the government.
Assange has been holed up in the Ecuadorian embassy in London for more than a year after he was granted asylum but denied free passage by British authorities out of the country.
He is wanted for questioning in Sweden over sex assault allegations but fears if handed over he will be passed onto the United States for his controversial diplomatic memo leaks.