They were arrested in July 2009 while hiking along the Iran-Iraq border.
All three, two in person and one in absentia, entered not guilty pleas during the five-hour closed-door hearing, according to their lawyer, Masoud Shafiei.
The third hiker, Bauer's fiancée, Sarah Shourd, is back in the U.S. after being freed from prison in September on $500,000 bail.
She has not responded to a court summons to return to Tehran and stand trial.
Iranian authorities said Shourd will be tried in absentia if she doesn't appear in court.
The three hikers claim they were hiking in Northern Iraq and inadvertently crossed an unmarked border into Iran. Their families say the Americans were just intrepid travelers out on a hike when they were arrested.
The U.S. government has denied the charges against them and demanded their release. Their lengthy 18-month detention has added to tensions between the two nations over issues like Iran's disputed nuclear program.
Iran, however, pressed forward with spy charges that could bring a maximum sentence of 10 years in prison if convicted.
Shourd and Bauer had been living together in Damascus, Syria, where Bauer was working as a freelance journalist and Shourd as an English teacher. Fattal, an environmental activist, went to visit them in July 2009 shortly before their trip to northern Iraq.
The Associated Press contributed to this report.