"The perpetrators of this crime must be severely punished. There will be no amnesty for such people," Ayatollah Ali Khamenei said on Monday, according to state media.
This was the first time that Khamenei, who has the final say in all major state matters, has commented on the poisonings that have worried parents across the country.
The first cases of mysterious illnesses were reported in November.
Since, hundreds of schoolgirls have been treated in hospitals across the country.
Schools for girls have almost been exclusively affected.
Parents and relatives are outraged and angry, especially as there is still no official explanation.
Iran's government believes the girls are being deliberately poisoned, perhaps by hardline groups opposed to girls' education.
Doctors have suggested some sort of toxic gas could be the source of the attacks.
Meanwhile, Iranian newspaper Entekhab and other local media reported that a journalist based in the the city of Qom, where the first cases of poisoning occurred, was arrested by authorities.
The journalist Ali Pourtabatabaei had been regularly covering the poisonings in Qom, a deeply conservative religious centre about a 150km south of Tehran that is especially important for Shia Muslims.
His arrest does not contribute to the "demystification of the rumours and news," the reform politician and journalist Abbas Abdi wrote on Twitter.
"I hope he will be released soon."
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