The protesters sang patriotic songs and chanted slogans against Croatia's centre-right government, which last week approved the Istanbul convention that was adopted by the Council of Europe in 2011, but still has not been ratified by Croatia's parliament.
Croatia's conservative opposition and the Catholic Church - the organisers of the protest - said they support combating violence against women and domestic violence, which is the main point of the convention, but are against its alleged introduction of "a third gender" into society.
"We are against gender ideology," Zeljka Markic, a leader of the conservatives, told The Associated Press.
"The definition of gender separated from sex was not agreed on the level of the European Union or on the level of the Council of Europe."
Since joining the EU in 2013, Croatia's population has been drifting toward the far right, including some who deny the Holocaust and have re-appraised the pro-Nazi Croatian Ustasha regime that ruled the country during World War II.
Top Croatian Catholic Church clergy have said before the rally attended by some 10,000 people that all those who support the Istanbul Convention should no longer be considered believers.
Speakers at the rally on Zagreb's Bana Jelacica square blasted the EU for allegedly wanting to "conquer" Croatia with its liberal policies. Several placards with photos of German Chancellor Angela Merkel were crossed out.
"They say we are backward because we believe in God," said Gordana Turic, a former ruling party politician. "The Istanbul convention is against the Christianity."
Several smaller counter-protests were set up along the route of the march, including one with a siren that tried to dampen the speeches by the conservatives on the main square. There were no major incidents.
Reporting by Associated Press, via Press Association.
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