The House of Representatives voted 98-42 against the legislation.
It was the first of four bills introduced to Parliament that aim to lift the country’s ban on same-sex marriages.
A separate bill was also being debated in the Senate.
Polls show that most Australians support gay marriage. But the Liberal Party-led conservative opposition coalition and many in the ruling centre-left Labour Party are against it.
Australian law defines marriage as a union between a man and a woman. Labour lifted its long-standing opposition to gay marriage last year, but Prime Minister Julia Gillard remains opposed to it.
In June, senators Boyce and Birmingham signed a Senate report calling for same-sex marriage. ”This situation is neither fair nor equitable,” they wrote.
Senator Wong and Senator Boyce were both critical of a speech on Monday by the Nationals senator Ron Boswell, who said he could ”not imagine a more savage attack on the family” and that all children had a right to a mother and a father.
Senator Wong said he was using the prejudiced language of the 1950s.