The charge of espionage is based on the aid workers listening to coast guard radio frequencies to find out where the smuggling boats with refugees were, The Guardian reports.
Several international human rights organizations have criticized the prosecution.
The defendants themselves believe that they did nothing but help people whose lives were in danger and deny any crime.
The events took place in 2018.
Amnesty International calls the accusations "father-like".
One of the indicted volunteers is the former Syrian refugee Sarah Mardini, 28, who came to Lesvos as a refugee herself in 2015. She and her sister then saved 18 refugees who could not swim from a sinking boat by pulling it ashore.
At the Rio 2016 Olympics, the sister competed for the Olympic refugee team in Rio 2016. Their story then inspired the Netflix movie "The swimmers".
Spent over three months in custody
Sarah Maridini and rescue diver Seán Binder, 28, were detained for over three months after they were arrested in 2018 on Lesbos.
More than four years later, the trial against them and the 22 other aid workers is being held.
“If I can be criminalized for mostly handing out bottles of water and smiles, so can anyone.
This trial is about the Greek authorities trying to crush compassion and prevent people from seeking safety.
But I trust that justice will prevail and that we will be able to continue with our lives, says Seán Binder before the trial according to Amnesty International.
The trial began on Tuesday in the town of Mytilini on Lesbos.
Negotiations continue on Friday.
Hear two of the accused aid workers in the clip above.