Speaking to students in Tehran on Saturday, Khamenei warned that both Israel and its primary supporter, the US, would be punished for their military action against Iran and "resistance groups" in West Asia, Xinhua news agency reported.
"The enemies... will undoubtedly receive a teeth-breaking response," he said.
He added that Iran was fully prepared with military, political, and logistical measures to counter "global arrogance" - a term Iranian officials use to refer to the US and its allies.
Khamenei also urged the Iranian people to fight against the "global arrogance" decisively without hesitation.
Referring to recent Israeli strikes on Gaza and Lebanon, Khamenei argued that US support for these attacks exposed "the hypocrisy of American human rights claims".
The Israel Defense Forces announced last week that it conducted "precise and targeted" airstrikes on targets in Iran in response to recent attacks from the country.
Iran's air defence headquarters said it successfully countered the Israeli attacks, which resulted in only "limited damage".
Iran and Israel have long been enemies, a rivalry that deepened following Hamas' attacks of October 7 last year and the subsequent Israeli invasion of Gaza. Israel has been battling Hezbollah in southern Lebanon, and overnight at least 19 people were injured in the central Israeli city of Tira after projectiles were launched over the border.
Khamenei's remarks come a week after Israel's latest round of strikes on Iran in response to the Islamic Republic's October 1 missile attack on the Jewish state, which itself was a response to the Israeli killing of leaders from Hamas and Hezbollah.