Federal leader Warren Truss argued such a move would breach a coalition promise to oppose gay marriage made before the last election.
The motion put by the Young Nationals was defeated by just 11 votes on Sunday.
Speaking against the motion Mr Truss said it was not just “about the issue of same sex marriage, it is also about honesty and integrity in government”.
“The Nationals have always respected the right of their colleagues to cross the floor and vote differently if they feel that is important to them,” Mr Truss told delegates in Bowral, in
the Southern Highlands.
“(But) for us to move away from the commitments that were made by our leadership prior to the last election would in my view be a breach in trust.”
Nationals MP John Williams said the motion was not necessary because “we have a conscience vote all the time in the National Party.”
“Barnaby (Joyce) has almost worn the carpet out crossing the floor,” he said.
“We vote where we like. We are free to vote how we like in the National Party.”
However, former Nationals MP Kay Hull, who in 2005 crossed the floor to vote against the Howard government’s legislation to sell off Telstra, said doing so was “no walk in the park.”
“It is a very serious issue, and you are not given the right to cross the floor automatically,” she told the conference.
A vote on two private members’ bills from government MP Stephen Jones and the Greens Adam Bandt is not expected until the end of the year, with supporters admitting they don’t yet have the numbers.
While Labor MPs have been granted a conscience vote on the issue, coalition MPs have not.
Mr Bandt has said he plans to use the parliamentary winter break to lobby MPs, and believes Opposition Leader Tony Abbott could be persuaded to grant those in the coalition a conscience vote.