The case centres on a 2020 operation where the Mare Jonio charity ship, operated by the Mediterranea NGO, picked up 27 migrants who had been rescued by a giant tanker in the Mediterranean Sea.
The activists then brought them to Italy.
The defendants include a doctor, the Mare Jonio's commander, and Luca Casarini, co-founder of the charity and a prominent left-wing activist. They all deny wrongdoing.
"This is the first indictment of its kind," lawyer Serena Romano told Reuters.
"All prior proceedings against NGO crews were shut down at the investigative stage or during preliminary hearings."
The charges come as Prime Minister Giorgia Meloni, who took office in 2022, continues her long-running campaign to reduce immigration flows across the Mediterranean.
The six defendants, five men and one woman, are accused of facilitating illegal immigration after they agreed to pick up the group of migrants, who had been stranded aboard the Danish tanker Maersk Etienne for more than a month.
At the time, neither the Maltese, Italian nor Libyan authorities had let the tanker bring the migrants ashore, according to Maersk Tankers, the operator of Maersk Etienne.
The group had been rescued from a sinking wooden dinghy near Malta as they sought to reach Europe.
The Mare Jonio took them to Sicily.
According to the Ansa news agency, prosecutors allege the rescue was financially motivated.
They cite a 125,000 euro ($A217,239) payment from Maersk to Idra Social Shipping, which owns the Mare Jonio. Mediterranea denies the accusation, calling the payment a "transparent donation" to support rescue efforts.
Casarini, who was a friend of the late Pope Francis, said the trial, ordered by a court in Ragusa, would offer an opportunity to scrutinise official conduct during the incident.
"We will call as witnesses the ministers and authorities who decided to leave 27 human beings adrift in the middle of the sea," he said.
Meloni's government, in power since 2022, has enacted policies aimed at reducing sea arrivals and limiting NGO rescue operations, though the case predates her administration.
Some members of the ruling coalition have accused the judiciary of looking to sink government efforts to prevent migrant crossings, including repeated rulings that have thwarted attempts to detain irregular migrants in Albania.
Earlier this year, several Mediterranea members, including Casarini, were targeted with spyware. The government has denied involvement and called for an inquiry.
