Khamenei, who has the final say in all Iran's national issues, said a "Zionist cancer" was gnawing into the lives of Islamic nations.
The annual Quds (Jerusalem) Day rally held across Iran on Friday, he said, was a "day of loud and clear shouts" against this "deadly cancer of Zionism which is gnawing into the lives of the Islamic nations".
The all-powerful cleric added that the "deadly cancer was spreading through the invading hands of the occupiers and arrogant powers."
His anti-Israel remarks came two days after the Quds Day rally during which President Mahmoud Ahmadinejad had also slammed Israel and reiterated his controversial belief that the Holocaust was a "myth."
Friday's pro-Palestinian rally was marred by massive protests against Ahmadinejad launched by supporters of those who stood against him in June presidential elections and who claim his re-election was rigged.
Ahmadinejad's anti-Israel comments enraged the global community, including key ally Moscow, just days ahead of his planned visit to the UN General Assembly meeting in New York.
On Sunday, Khamenei said attempts by Israel, Western powers and foreign media networks to weaken Iran had failed.
"The enemies tried to undermine the Quds Day rally, but the rally showed that the schemes of the enemies were not effective," the cleric said as worshippers attending his sermon at Tehran university chanted, "Leader, we offer our blood to you!"
He particularly lashed out foreign media networks, saying they were "poisoning the atmosphere in Iran."
"In the past few months, Western leaders fell for their media, professional press analysts and radios and televisions and thought they could influence the Iranian nation. But you showed that they were chasing a mirage," he told the crowds of worshippers.
"This year, more than before, they tried to weaken the Quds Day, but the glorious Quds Day in Tehran showed the whole world the direction in which the revolution and Iran was heading.
"It showed that their (Western politicians) tricks, spending money and political evilness does not influence the Iranian nation."
Khamenei's sermon marking Eid and the end of the fasting month of Ramadan was attended by Ahmadinejad and other top officials of Iran, including opposition supporter Akbar Hashemi Rafsanjani and parliamentary speaker Ali Larijani.
Mohsen Rezai, former head of Iran's elite Revolutionary Guards and one of the defeated candidates in the June 12 presidential election, was also present during the sermon.
