The rally, held in front of the city hall under steady rain and watchful police supervision, brandished slogans decrying "hate-filled" rhetoric seen as encouraging violence against gays and lesbians.
"It's very important for us all to be here… to show an image of love in dignity," said one of the demonstrators, Wilfried de Bruijin, a Dutch gay man living in France who was brutally attacked in Paris last weekend while walking with his boyfriend.
Photos of de Bruijin's battered face have been shared on social networking sites thousands of times.
The protest came after France's Senate earlier Wednesday passed the first article of a bill that clears the way for a law enshrining the right for homosexual couples to marry.
The issue has stirred vehement debate in France, an officially secular but predominantly Catholic nation that has a long history of tolerance towards many varieties of sexuality as long as they are kept discreet.
The bill has galvanised public opinion in France, not only on the streets where supporters and opponents have regularly faced off in nationwide protests attracting hundreds of thousands, but also among friends and family.
Despite this, it has made its way through the legislative process, adopted by the cabinet in November and the parliament's lower house in February. It is now in the Senate, where debate on the bill is due to end Thursday or Friday.
"It's difficult to say whether there are more homophobes than before, but there are more who are expressing themselves," said Nicolas Gougain, spokesman for the Inter-LGBT, a rights group for lesbians, gays, bi and trans-sexuals.
Protests have been led mainly by religious groups and conservatives in a country that is officially secular but predominantly Catholic.
President of gay-rights group SOS Homophobie, Elizabeth Ronzier is clear who is to blame for the rise in reported homophobic incidents recorded by her organisation in recent months.
“We have seen a thirty percent rise in the number of homophobic incidents since October. This is a result of the opposition towards the gay marriage bill," Ronzier told The Local.
“These people say they are not homophobic but they are. Homophobia has become trivialized, which is proved by the number of verbal assaults on gay people, which often to lead to physical assaults."
In January, hundreds of thousands of demonstrators flooded into Paris for an anti-gay marriage march. Last month, police fired tear gas on people protesting the bill, and dozens were arrested.
Opponents said Wednesday they would organise another mass protest in Paris on May 26 if the law is approved, to demand its withdrawal and a referendum on gay marriage.
Polls in France regularly show that a majority of people support gay marriage, but far less are in favour of same-sex adoption.