Hours later, Israeli Prime Minister Benjamin Netanyahu used his first press conference since the start of the war to make his own strong statement, issuing a veiled threat to kill Khamenei and defending the military assault on Iran.
"I wouldn't issue life insurance policies on any of the leaders of the terrorist organisation ... I don't intend to give an exact message here about what we are planning or what we are going to do," Netanyahu said, standing between two Israeli flags and taking questions via video link.
Netanyahu said Israeli attacks had killed a top Iranian nuclear scientist and hit others.
In Iran, Khamenei did not appear in person and his remarks were read out by a state television presenter.
No images have been released of him since an Israeli strike at the start of the war that killed much of his family, including his father and wife.
Khamenei's statement called on Iran's neighbours to shut US bases on their territory and warned Iran would continue to target them.
"I assure everyone that we will not neglect avenging the blood of your martyrs," said the hardline cleric, who is close to Iran's top military force.
"The popular demand is to continue our effective defence and make the enemy regret it. The lever of blocking the Strait of Hormuz must continue to be used," Khamenei added, referring to the shipping route through which a fifth of global oil normally passes along Iran's coast.
Iranian officials have said Khamenei was lightly wounded in the initial February 28 airstrikes, but the extent of his injuries is unclear.
Rescue efforts were under way after a US Air Force refuelling plane went down in Iraq, the US military said.
The aircraft is part of the American military operation against Iran, but the crash was not due to hostile fire or friendly fire, the military said.
Two tankers were ablaze in an Iraqi port after a hit by suspected Iranian explosive-laden boats, a clear sign of defiance toward US President Donald Trump, who said on Wednesday the United States had already won the war.
Images verified by Reuters as filmed from the Iraqi port of Basra showed ships engulfed in massive orange fireballs that lit up the night sky. At least one crew member was killed.
Hours earlier, three other ships were struck in the Gulf. Iran's Revolutionary Guards claimed responsibility for at least one attack - on a Thai bulk carrier that was set ablaze.
Another container vessel reported being struck by an unknown projectile near the United Arab Emirates.
On another front in the unpredictable war, Israeli airstrikes hit a building in central Beirut on Thursday, sending thick smoke above the Lebanese capital.
Israel also ordered residents out of another swathe of southern Lebanon, intensifying its offensive against the Iran-backed Hezbollah group after it fired its biggest volley of rockets into Israel since the start of the war.
So far the war has killed more than 2000 people, including almost 700 in Lebanon.
Trump played down higher oil prices resulting from the conflict.
"The United States is the largest Oil Producer in the World, by far, so when oil prices go up, we make a lot of money," Trump wrote on social media.
US Treasury Secretary Scott Bessent told Sky News that America's navy, perhaps with an international coalition, would escort vessels through the Strait of Hormuz when it is militarily possible.
Separately, Trump said the Iranian men's national soccer team was welcome to participate in the 2026 World Cup, which the US is co-hosting, but added it was not appropriate that they be there "for their own life and safety".
Inside Iran, residents said security forces were increasing their presence to demonstrate continued control.
"Security forces are everywhere, more than before. People are afraid to come out, but supermarkets are open," teacher Majan, 35, said by phone from Tehran.
with AP
