Pink Dot has been held every year since 2009. Attendees wear pink clothing and sit down for a mass picnic that ends with the forming of a pink dot.
But this year it falls on the eve of Ramadan, prompting an Islamic teacher to start a Wear White campaign against homosexuality, which has been supported by a Christian organisation.
Gay sex is illegal in Singapore.
Government leaders, as well as national organisations representing mosques and churches, have in recent weeks called on both religious and gay rights groups to act with restraint.
The Pink Dot rally proceeded peacefully on Saturday evening with no sign of anti-gay campaigners. Organisers said 26,000 people attended the event.
Wear White issued a statement saying it discouraged supporters from attending Pink Dot, as "it should be an event that no Muslim is associated with".
Some chose to go online instead to protest at the event.
About 4,000 people so far have taken part in a virtual rally called FamFestSG. Its Facebook page carries a quote from Prime Minister Lee Hsien Loong that defines the term family as "one man one woman marrying, having children".
The backlash has reignited the issue of gay rights in the largely conservative city-state.
In recent years, there have been two legal attempts to declare that the law banning gay sex is unconstitutional.
Singapore's government has said in the past that while it will retain the law to reflect mainstream society's stand on the issue, it will not be actively enforced.

The 23-year-old joined QPR from Swindon in May but he has only made three appearances for the Championship club.
Mark Cooper's League One side have been hit by several injuries this season.
"An injury has meant he's subsequently struggled for game time," said QPR's director of football Les Ferdinand. "He's only away for a month. He's still got a massive future at QPR."
Find all the latest football transfers on our dedicated page.

An Action on Hearing Loss Cymru survey found many job seekers are reluctant to list it on job applications.
The charity spoke to one deaf person who had not had an interview in 16 years of stating they were deaf on CVs.
Many public sector organisations were found to show poor practice in the report to mark Deaf Awareness Week.
Maggie Hampton of Fairwater, Cardiff, told BBC Radio Wales that she found it "very disheartening."
In the interview she said: "It was very very difficult. Nobody wanted to employ a deaf person. Even in the most lowly of jobs really. I never got interviews. I just never heard back.
"I did follow it up with one organisation and asked 'why have I not got an interview?' And the reply was 'Well you are deaf, you couldn't possibly manage.'
"So I did cleaning for two or three years, it was the only thing I could do.
"I didn't like it very much but I did it because I needed to work.
"It was very hard and very disheartening. Just that feeling that nobody wants you. And that feeling that you cannot contribute anything."
She  said many deaf people do not declare the fact on their CVs as they "want to fit in".
Action on Hearing Loss Cymru director Richard Williams said 84,000 people, or 4% of Wales' working-age population, are deaf or wear hearing aids.
"That's the equivalent to filling the Millennium Stadium with people who are all being discriminated against at work because they can't hear well," he added.
An annual gay rally in Singapore has drawn thousands amid an unprecedented backlash from religious groups.
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Swindon Town have re-signed midfielder Ben Gladwin on a 28-day emergency loan deal from Queens Park Rangers.
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Fear of discrimination means 84,000 deaf and hard of hearing people hide their condition from prospective employers, new research has revealed.