The protests were accompanied by a 24-hour strike -- the biggest yet since the disaster -- this time called by Greece's leading private as well as public sector unions, France24 reported.
Clashes erupted at Syntagma Square near parliament in Athens, where police fired tear gas and stun grenades at demonstrators hurling firebombs and rocks.
As protesters retreated, they smashed traffic lights and shop windows and set rubbish bins on fire, AFP reporters said.
A plainclothes police driver for a leftist MP was lightly hurt a demonstrator smashed his car window, said state television ERT.
Ten people were detained for questioning, police added.
The February 28 train crash exposed decades of safety failings in Greek railways and has put major pressure on the conservative government ahead of national elections.
Police said 25,000 people protested in Athens on Thursday, as well as around 8,500 in each of the country's next largest cities, Thessaloniki and Patras, where brief clashes also broke out, police said.
Thursday's industrial walkout shut down the civil service, flights and ferries.
Many protesters urged the government of Prime Minister Kyriakos Mitsotakis to resign over what is the country's deadliest rail accident.
"This crime will not be forgotten," demonstrators from the country's communist union PAME chanted as the crowd marched towards parliament and the offices of rail services company Hellenic Train in Athens.
Students shouted "murderers" and marchers threw flyers of Mitsotakis wearing a stationmaster's cap, captioned "it's everyone's fault but mine".
The rail disaster occurred shortly before midnight when a passenger train crashed head-on into a freight train in central Greece after both were mistakenly left running on the same track.
Most of the passengers were students returning from a holiday weekend.