Wednesday's clashes erupted over the detention of the three activists, arrested on charges of hooliganism and beating a state television reporter.
Scores of protesters rushed to a police station where the detainees were held after one slipped out a note saying they had been beaten and abused by police.
Thousands of people then flocked to the parliament building to demand the release of the detainees.
Police armed with truncheons tried to beat back the demonstrators, some of whom were wielding sticks.
Opposition supporters said dozens of protesters were injured and hospitalised.
|Moscow has complained bitterly about Nato's military exercises in Georgia [AFP]
Eka Zguladze, the deputy interior minister, said 22 protesters, six police officers and a journalist were injured, and that none of the injuries were life-threatening.
Authorities freed the activists early on Thursday, citing an appeal from the head of the Georgian Orthodox Church.
The television pictures of bloodied protesters appear to have fueled tensions just as public interest in the protests, which began early last month, had appeared to have been waning.
Matthew Collin, Al Jazeera's correspondent in Tbilisi, said: "There's certainly a deep sense of anger among opposition activists here in the Georgian capital.
"Last night's clashes have simply made them more determined to drive president Saakashvili out.
"The protests had started to run out of energy, with fewer attending the rallies, but there are now fears that the confrontation could escalate."
Moscow has complained about Nato's military exercises, cautioning the West against helping Saakashvili rebuild Georgia's army, and has denied any involvement in the rebellion at the army base.
Opposition leaders called the mutiny incident a charade cooked up by Saakashvili to rally support amid pressure from the protesters.
There have been isolated incidents of violence during the demonstrations, but the authorities have vowed not to interfere as long as protesters do not instigate violence.
A violent police crackdown on similar protests in 2007 damaged Saakashvili's reputation and prompted some former allies to join the opposition.
Saakashvili was in Prague on Thursday for a summit of some former Soviet states.