The demonstration gathered up to 10,000 people according to journalists' estimates and was held two days after conservative Prime Minister Andrej Plenkovic's government sent the treaty for ratification by the parliament.
The Council of Europe convention -- the world's first binding instrument to prevent and combat violence against women, from marital rape to female genital mutilation -- has split the country.
The Balkan nation's conservatives, supported by the Catholic Church along with hardliners from the ruling HDZ party, oppose the ratification.
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They argue that under the guise of protecting women the so-called Istanbul Convention promotes what they call a "gender ideology" which would undermine the traditional family.
"I'm a father not a parent!" and "Halt violence against Croatia!" read some of the banners carried by the protestors who gathered in front of the HDZ seat before marching through central Zagreb.
The demonstrators, who also came from other parts of the country, waved Croatian flags, prayed and sang patriotic songs.
"We feel threatened in our own country. The Istanbul convention is against Christianity," Gordana Turic told the crowd as they stopped at the main square.
The protestors chanted 'Treason, Treason' when Plenkovic's name was being mentioned and the organisers urged MPs not to ratify the treaty.
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"I want family and family values preserved while the convention is against them," Ivana Horvat, a 38-year-old administrator, told AFP.
In a bid to calm down tensions, Plenkovic, representing moderates within HDZ, amended a bill ratifying the treaty.
It includes a statement saying notably the treaty does not imply the need to "introduce gender ideology" in Croatia's schools or change the constitutional definition of marriage as a union between a man and a woman.
In the past weeks, the Catholic Church launched a fierce campaign against the convention's ratification with priests publicising the protest after masses.
"The time has come to get out on the streets," Bishop Vlado Kosic said on Facebook urging Christians to join the protest.
He earlier labelled the convention a "plague".
Nearly 90 percent of Croatia's 4.2 million people are Roman Catholics and the Church plays an important role in society.
The convention has so far been ratified by 28 countries, including 17 European Union member states.
Croatia became the bloc's newest member in 2013.
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