Some 100 members of the conservative-dominated parliament complained to chief prosecutor Gholam Hossein Mohsen Ejeie that Mousavi had committed a "national crime" by alleging fraud in the June vote, state media reported.
"The crime committed by Mousavi is a national crime and he has violated the rights of the Iranian people," the official IRNA news agency quoted one of the MPs, Hamid Rasaie, as saying.
The demand for Mousavi's prosecution came a week after a similar call for legal action against Ahmadinejad's other moderate challenger, former parliament speaker Mehdi Karroubi.
Rasaie said the letter of complaint handed to the prosecutor was accompanied by "concrete evidence against Mousavi."
"We were hoping that Mousavi would change course and keep his distance from movements that follow the same line as our enemies but we have seen no change in his activities," the MP said.
"His actions have harmed the reputation of the regime."
Both Mousavi and Karroubi refused to drop their allegations of fraud in the June 12 election as the official results giving Ahmadinejad brought hundreds of thousands onto the streets in Iran's worst unrest since the 1979 revolution.
The protests sparked a massive crackdown in which 36 people died, according to official figures, and 72, according to the opposition. A further 4,000 people were detained of whom some 140 have been put on trial.
In the latest judgement from one of the hearings, Iranian-US scholar Kian Tajbakhsh was sentenced to more than 12 years in jail after prosecutors told a Tehran revolutionary court that he was involved in "spying" and "acting against national security."
"I can not reveal the exact term, but I can say it is more than 12 years," IRNA quoted defence lawyer Houshang Azahari as saying.
Prosecutors charged that Tajbakhsh was "acting against national security by being a member of an internet network linked to the CIA (US Central Intelligence Agency)," IRNA said.
Tajbakhsh was also accused of "provoking people during the riots after the election, and of having connections with foreign elements who are known to be anti-Iran," it added.
"He was agitating for the overthrow of the regime."
In his own testimony to the court, Tajbakhsh said that he was a "victim of the American programme of softly toppling the regime in Iran."
It is not the first time that the academic has been in custody in Iran. In 2007, he was detained while working for the Soros Foundation.
The lengthy jail sentence came despite pleas for his release from both the United States and Canada.
On September 25, US Secretary of State Hillary Clinton and Canadian Foreign Minister Lawrence Cannon jointly urged Iran to release several foreigners, including Tajbakhsh, as a "humanitarian gesture."
On Saturday, Iran released on bail Newsweek journalist Maziar Bahari, who was also arrested during the post-election unrest.
The Iranian-Canadian, who had been living in Iran for 10 years, flew out of the country and arrived in Britain on Tuesday, his employer said.
"We are delighted to announce that Newsweek journalist Maziar Bahari has arrived in London in time to witness the upcoming birth of his first child," the magazine said.
The semi-official ILNA news agency reported on Saturday that bail of three billion rials (300,000 dollars) had been posted for Bahari's release.
The news agency said the journalist was accused of sending false election reports, disrupting public order by participating in illegal gatherings, and holding classified documents.
Newsweek rejected the charges and repeatedly demanded his release.
