Sam Rainsy, president of the Cambodia National Rescue Party, and his deputy Kem Sokha said they had done nothing wrong and would contest the charges of "inciting civil unrest".
"The prosecutors have no evidence whatsoever," opposition member Mu Sochua said outside the Phnom Penh Municipal Court.
Protests for a higher minimum wage for some 500,000 garment workers culminated in a clash with police Jan 3, 2014 Five protesters were killed and dozens injured after police opened fire on rock-throwing strikers.
Sochua said there should be an independent investigation into the use of force by police.
The party has been calling for long-time Prime Minister Hun Sen to step down after what it alleges were fraudulent elections in July. The party has strong links to the country’s labour unions.
Rainsy and Sokha arrived at court Tuesday morning to cheers and applause from around 2,000 supporters gathered outside.
The crowd turned out in defiance of a ban on public gatherings of 10 or more people announced by the government after Jan 3.
Some held signs asking the United Nations for help and welcoming UN special envoy to Cambodia Surya Subedi, who is in Cambodia this week on a fact-finding mission.