Backed by religious groups, the demonstrators marched across the city to voice their opposition to a bill that aims to comply with a 2019 court ruling in favour of LGBTQ rights.
Some held signs reading "we say no homosexuality" and "protect our children".
The bill "would open floodgates of immorality and abomination", said Pastor Pulafela Mabiletswane Siele of the Evangelical Fellowships of Botswana, a Christian group, delivering a petition to the parliament.
"We urge our parliament to opt for a referendum regarding this matter," Siele said.
The protest comes amid a wider pushback against LGBTQ in Southern Africa.
Lawmakers in Namibia have backed legislation to ban same-sex marriage and punish its supporters, following a Supreme Court ruling that allowed for the recognition of some unions contracted abroad.
Botswana's High Court in 2019 ruled in favour of campaigners seeking to strike down jail sentences for same-sex relationships, declaring the punishment to be unconstitutional.
The government sought to revoke the ruling but lost in appeal in 2021.
Gay sex had been banned since 1965 in the socially conservative African nation, where offenders could face up to seven years in prison.
The bill that is expected to be discussed in the coming weeks removes the related provisions from the penal code.
Receiving the petition on behalf of parliament, opposition lawmaker Wynter Mmolotsi said MPs would take into consideration the views of the church.