Khamenei also said that "resistance" was the only way to save Palestine and called for the Israeli leaders behind the recent assault on Gaza to be put on trial.
"The political and military leaders of the occupying regime that played the role in the disaster of Gaza must be put on trial. This is the first step that should be taken," Khamenei told the conference.
"The only way to save Palestine is resistance.
"Support and help to Palestinians is a mandatory duty of all Muslims. I now tell all Muslim brothers and sisters to join forces and break the immunity of the Zionist criminals."
Khamenei's comments are likely to disappoint the new US administration, which has indicated it is keen to break with its predecessor and engage Iran if Tehran will "unclench its fist".
Obama has suggested that he would be prepared to engage Iran on a range of issues, including the country's nuclear ambitions.
Khamenei, who has the final say on all policy in Iran, has so far not directly addressed the issue of Obama's overtures but has in the past said US governments could not be trusted.
The US has long called on Iran to end its support for organisations like Lebanon's Hezbollah and the Palestinian Hamas faction, both of which Washington considers "terrorist" organisations.
Iran says that Hamas is fighting an illegal occupation by Israel on Palestinian land.