The final vote in the Senate was 41 to 26 after the bill was rejected Wednesday in the House of Representatives, the Australian Broadcasting Corp. reported.
The lower house vote was 98 to 42.
The bill had been introduced in both chambers by members of Australia's ruling Labor Party.
The overwhelming rejection has caused other supporters of same-sex marriage to consider new tactics.
Liberal MP Warren Entsch said he has written a "civil partnerships" bill that he thinks might have a better chance of passage than those that change the nation's marriage laws.
"It's two persons who are in a relationship as a couple regardless of their sex and who meet the eligibility criteria," he said.
The Liberal Party has an official policy defining marriage as a union between a man and a woman.
Greens MP Adam Bandt rejected the idea of civil unions, saying they would be "a step backwards."