Under a forest of yellow flags emblazoned with black lions, Flemish separatists dominated the procession through the European Union's capital, which marched from Parc du Cinquantenair near the European Commission's headquarters towards the European Parliament.
The crowd, which marched under a banner proclaiming "Europe, we'll vote for self-determination", numbered almost 3,000 people, according to a senior police officer.
Draped in their regional colours — red and yellow — the Catalans chanted in their regional language for "independence".
Marta Giralt, a 38-year-old translator, came from Barcelona to "defend the right of the Catalans to vote" in a referendum on independence from Spain.
Regional leader Artur Mas has set the vote for November this year but Madrid has declared it illegal.
"We want to make our voices heard and expand ourDemonstrators hold Catalan separatist flags at the Pedralbes Palace in Barcelona on October 23, 2013 movement," Girault told AFP.
Small groups of Scots, who are due to vote in a referendum on independence from Britain in September, also stood alongside South Tyroleans, Lombards, Corsicans and a handful of Bretons.
The demonstration was organized by the International Commission of European Citizens, an body which represents the main separatist movements in Europe.