The Australian Christian Lobby has called on the ACT government to remove rainbow flags from public land, claiming they are political.
But a defiant chief minister Andrew Barr has vowed the distinctive emblems will fly again.
Mr Barr said on social media last week the flags would go up for a week from Valentine's Day to support marriage equality.
"Canberra is Australia's most LGBTI friendly city," Mr Barr said in a video.
"We want to celebrate that and show our support for marriage equality."
ACL managing director Lyle Shelton said Mr Barr's reasons proved the motive was political.
"If the ACT government is going to make it open for political causes to have their flags flown at public expense on public land, there has to be equal preference for all political causes," Mr Shelton said.
"This is a flag that represents a political cause that's very prominent in the political discourse at the moment.
"That political agenda is not something that all Australians have signed onto.
"Things like safe schools and teaching children their gender is fluid, I think many Australians believe that if marriage is going to be redefined there should be a public vote before the flag of a political movement is displayed on public land."
Mr Shelton said he accepted the ACT government had a policy position in support of marriage quality.
"But this is public land in the parliamentary triangle, which belongs to all Australians not just Canberrans, and it's being used to push a political agenda," he said.
"I think most Australians would expect that to be an apolitical space."
In response, Mr Barr said Mr Shelton's "conservative views on this issue" were well known and he promised the flags would fly again during the spring LGBTIQ festival.
"The vast majority of Canberrans support marriage equality and want the Federal Parliament to do its job and legislate as such," he said.
"The ACT government will continue to advocate on this issue and on the rights of LGBTIQ people more generally because it is the right thing to do.
"Last year the Assembly voted the ACT should do what it can to support same sex marriage.
"A few days ago, as these beautiful flags flew over the capital, the ACT Assembly passed legislation to automatically recognise same sex unions from overseas."