Lawrence Khong, founder and pastor of the 10,000-member Faith Community Baptist Church, has been the most vocal critic of homosexuality and the Pink Dot rally.
In a statement, he said he could not understand why authorities were allowing the rally to take place.
"I find it even more disconcerting that the event is being used as a platform of public persuasion to push their alternative lifestyle," he said. "I would like to see our government leaders draw a clear line on where they now stand with regard to this moral issue."
Deputy Prime Minister Tharman Shanmugaratnam said he believed Singaporean society should be one "where you don't go pushing your own beliefs and preferences, but at the same time everyone else keeps the balance in society and avoids creating conflict."
Former lawmaker Siew Kum Hong, who tried to get Parliament to repeal Section 377A unsuccessfully, said he believed that the legislation will be overturned eventually.
"I've always maintained that the government's position is untenable. When presented with a chance to repeal 377A, it decided to avoid making a principled decision and instead opted to kick the can down the road."
Other opposition came from an Islamic teacher who encouraged Muslims to wear white Saturday on the eve of the holy Muslim month of Ramadan, which was interpreted as a response to a Pink Dot video showing a Singaporean Muslim declaring his support for the LGBT community.
The LGBT supporters wore pink in the rally, whose highlights include large crowds standing together with pink torchlights at night, creating a spectacular aerial view.