He said 700 of Mousavi's supporters have been arrested, the former candidate's communications offices have been ransacked, and Mousavi himself has come under government restrictions.
Watch "Good Morning America" Saturday, June 20, for more of ABC News' interview with Mohsen Makhmalbaf.
Makhmalbaf said Mousavi has been "assigned" a government protection force that strictly controls his movements and access to the media. "Mousavi is 20 percent free and 80 percent under house arrest," he said.
Ahmadinejad was in attendance at Khamenei's speech, but Mousavi was not. Many read this as a sign the men were made aware of the supreme leader's stance ahead of the speech.
Addressing the protestors, Khamenei said, "It's a wrong impression that by using street protests as a pressure tool, they can compel officials to accept their illegal demands. This would be the start of a dictatorship."
He challenged opposition supporters over charges of vote-rigging, saying, "Iran's laws do not allow vote-rigging. ... With these laws, how could it be possible to have such vote-rigging?"
Mousavi has appealed for the election result to be annulled.
Supporters of the government, carrying posters of Khamenei, Ahmadinejad and the late Ayatollah Ruhollah Khomenei, the father of the 1979 Islamic revolution, packed streets outside the university.
"Ahmadinejad has been our president for four years, and during this time he has always told the truth to our people," Javid Abbasirad, 48, told Reuters, as he stood outside the university gates.
At the same location, hundreds of university students had protested against the election results Sunday, hurling stones at riot police. Tehran University was the site of serious clashes against student-led protests in 1999 and is one of the nerve centers of the pro-reform movement in Iran.
Today, some members of the crowd were draped in Iranian flags, others carried placards and flyers with anti-Western slogans. "Don't let the history of Iran be written with the pen of foreigners," one flier said.
On Thursday, thousands of anti-government protesters were joined by Mousavi in a march to mourn the deaths of at least eight demonstrators killed in the wake of last week's elections.
The protesters, seen on TV, many wearing black and holding photos and the names of those killed, jammed Iman Khomeini square, according to various media reports. "Why did you kill our brothers?" read one sign, Reuters reported, "Our martyred brothers we will take back your votes," read another.
Foreign media are still officially barred from covering unauthorized demonstrations.
With many foreign reporters now forced to leave the country, the protestors inside Iran have become the world's eyes and ears on the ground, filming protests on their cellphones and distributing the footage on Web sites such as Twitter, Facebook and YouTube.
Facebook users who speak Persian [Farsi] helped create a Persian-language translation of the site, which was launched Friday. Google also added Farsi to its Google Translations tool today.
"We feel that launching Persian is particularly important now, given ongoing events in Iran," the company said in a release.
Reuters and Associated Press contributed to this report.