Two gunmen opened fire at the National Bardo Museum in an operation claimed by the Islamic State group.
Some 21 detained suspects, including two women, attended Tuesday morning's unannounced hearing, defence lawyer Samir Ben Amor said.
Three others, who were not under arrest, were not present, he said.
Tunisians protest for right not to fast during Ramazan
About 30 people are also on trial in absentia, defence lawyer Rafik Ghak said.
The suspects were not named.
Since its revolution in 2011, Tunisia has faced a series of militant attacks that have claimed the lives of more than 100 soldiers and police along with 20 civilians and 59 foreign tourists, according to an official tally.
A month after the Bardo attack, 38 foreign holidaymakers including 30 Britons were killed in a gun and grenade attack on a beach resort near the city of Sousse.
That November, a suicide bombing in the capital killed 12 members of the presidential guard.
IS claimed all three attacks.
Gunmen attack UN convoy in Libya, briefly hold staff
French lawyers for the victims of the Bardo attack and their families have said an investigation had left "several dark areas".
One of them, Philippe de Veulle, has said he will boycott the trial, saying it would not offer "independent justice".
Some 26 people went on trial in May over the Sousse attack, including six security personnel accused of failing to assist people in danger.
More than 5,000 Tunisians have travelled abroad to join militants groups, mainly in Iraq, Syria and Libya.
COMMENTS
Comments are moderated and generally will be posted if they are on-topic and not abusive.
For more information, please see our Comments FAQ