The comment represents the first, direct call for Trump's death by an emcee at the funeral, which has seen posters and graffiti calling for the killing of Trump and Israeli Prime Minister Benjamin Netanyahu.
That's been a hard-line demand even as Tehran negotiates with the US over a permanent end to the war that's disrupted global energy supplies.
Mohammad Rasouli, a poet, drew calls of "Death to America!" and "Death to Israel!"
Speaking to the crowd over loudspeakers at the funeral on Sunday, Rasouli asked, referring to Trump: "Why is the most bastard man in the world still alive?"
The question drew cheers from the crowd.
A far-larger crowd for the funeral than the day before attended the ceremony on Sunday.
Mourners dressed in black walked to the site, carrying banners and flags honouring Khamenei and also calling for Trump's killing as the American president gave a speech in Washington for the 250th anniversary of the founding of the US.
"We've had tremendous success," Trump said, speaking of the American military.
"You look at Venezuela, you look at Iran. We wiped it out, wiped out their military."
The funeral for Khamenei, who ruled Iran for decades before he was killed at age 86 in a February 28 airstrike in the opening moments of the Iran war, could provide a boost for the country's theocracy and its new supreme leader, his son Ayatollah Mojtaba Khamenei.
That is important as Iran tries to leverage its hold on the Strait of Hormuz in negotiations with the US over a permanent end to the war and as concern lingers that Israel could attack again.
The funeral was delayed as the war raged, and talks are on hold until it is over.
