Carrying Georgian and Ukrainian flags and placards that read "Stop Putin!", the protesters gathered on the city's main thoroughfare, following the call of the former president Mikheil Saakashvili's United National Movement party (UNM).
Saakashvili addressed the rally by video link from Kiev as the cheering crowd chanted his name.
"Let's show Georgia's government that the nation is united against the serious threat to its independence, its future," he said.
Saakashvili's UNM party has accused the ruling Georgian Dream coalition government of not confronting what it claims is Russia's creeping annexation of Abkhazia and South Ossetia.
Moscow officially recognised their independence after fighting a five-day war with Georgia in August 2008.
After that war, Moscow stationed thousands of troops in the two separatist statelets in a move condemned by Tbilisi and its Western allies as an illegal occupation.
Russia has recently made further steps to integrate the tiny regions and has offered them "alliance and integration" agreements that would dramatically strengthen their ties with Moscow.
The move was condemned by the Georgian government as "yet another step against Georgia's sovereignty and territorial integrity."
Georgian Dream, assembled by former prime minister and billionaire tycoon Bidzina Ivanishvili, came to power in 2012 and ended a decade-long rule of staunchly pro-Western Saakashvili and his UNM party.
Scores of former top Saakashvili officials have been arrested over the last two years for alleged wrongdoing, prompting warnings from the West over selective justice and persecution of political opponents.
Accused by prosecutors of abuse of power, Saakashvili lives in a self-imposed exile in the United States.
