But police denied the demonstrators from the Inter-faith Coalition against Homosexuality permission to march through the capital Kampala for "security reasons".
Gathered on a rugby field outside the east African nation's capital Kampala, the roughly 100 anti-gay activists displayed dozens of placards calling for the arrest of gays and lesbians.
"We are fighting against the fresh campaign for homosexuality and lesbianism in this country," said organiser and pastor Martin Sempa. "Homosexuality and lesbianism break three laws; the laws in the Bible and the Koran, the laws of nature and the laws of the land, the Ugandan Constitution."
Ugandan law punishes sodomy with life imprisonment.
The protest was a response to a call from a gay and lesbian advocacy group, the Sexual Minorities Groups in Uganda (Smug), which for the first time held a news conference demanding recognition a week ago.
Most hiding their faces behind masks at that news conference, the gays also accused the police of harassment.
Tuesday's protesters carried dozens of placards ranging from "Arrest all homos" to "God loves homos, he hates homosexuality".
Other placards called for the sacking of Katherine Roubos, a U.S. intern at local independent newspaper Daily Monitor, for reporting on the experiences of gays in Uganda.
"Aga Khan, fire Katherine Roubos, homo propagandist," one said, while another read: "Government deport Roubos."
Daily Monitor is part of the regional Nation Media Group partly owned by the Aga Khan, spiritual leader of more than 15 million Shia Ismaili Muslims worldwide. He is visiting Uganda -- a mainly Christian country with a Muslim minority.
Roubos denied campaigning for gays, saying she was simply doing her work. "I was assigned a story by the editor and I did it objectively. My job is to report on events, not my personal opinions," she said.
Paul Kagaba, a gay member of Smug, criticised the protest.
"We did not hold the press conference against churches and government. We were simply telling everyone to leave us alone and today's protest is clearly misplaced," he told Reuters.
Activists say Uganda, with a population of 31 million, has some 500,000 gays and lesbians, who face much discrimination.
Police spokeswoman Judith Nabakooba said they denied the marchers permission through the city centre for security reasons. "We could not guarantee their security in the middle of town and their request was at short notice," she said.