More than two dozen aid workers will go on trial over their role in helping migrants reach Greece between 2016 and 2018 on Thursday.
The defendants face a raft of charges, including espionage, forgery and the unlawful use of radio frequencies.
Human rights groups have condemned the trial as being politically motivated and Amnesty International has called the charges “farcical”.
Lesbos, the island at the centre of the migration crisis, will host the trial.
Among the 24 defendants, 17 of whom are foreign nationals, is Syrian refugee Sarah Mardini, the sister of the Olympic swimmer Yusra Mardini. The siblings came to international attention in 2015 after dragging their refugee boat to safety when the engine failed.
Ms Mardini has been barred from entering Greece to defend herself and has been forced to watch the trial from Germany, where she was granted asylum.
Prosecutors allege that the aid workers, who were affiliated with a search and rescue group, monitored the Greek Coast Guard’s radio channels and used a vehicle with a fake military licence plates to enter restricted areas on Lesbos.
If found guilty they could face five-year prison sentences.
Some also remain under investigation for a number of other felonies, including human smuggling, and if convicted could face further sentences of up to 25-years.
Sean Binder, an Irish aid worker facing trial, told the Irish broadcaster RTÉ: “I am happy to defend myself, I know I did nothing wrong, and we can prove that.”
“I’m being charged with crimes that I am supposed to have committed a year before I was ever on the island,” he added.
Nils Muižnieks, Director of Amnesty International’s European Regional Office, said in a statement: “The charges they face are farcical and should never have come to trial”.
“This emblematic case demonstrates how far the Greek authorities will go to deter people from helping refugees and migrants. Stopping rescue operations doesn’t stop people from making dangerous journeys, it simply makes those journeys more perilous,” he added.
The trial comes as Greece’s conservative government, led by Kyriakos Mitsotakis, has clamped down on human rights groups in an attempt to limit the number of migrants entering the country.
Last year it introduced tougher rules for the operation of dozens international aid groups, which critics say makes it harder to operate in the country.