Addressing the gathering in Tehran for the first time since 2012, Ayatollah Ali Khamenei said Mr Trump had a "poisonous dagger" which he would "push into the nation's back".
He also said the outpouring of grief at the funeral for Iran's top general Qasem Soleimani, who was killed in a US airstrike earlier this month, shows that Iranians support the Islamic Republic.
He added that America had killed the most effective commander in the fight against the Islamic State group in a "cowardly" manner.
Ayatollah Khamenei last delivered a Friday sermon in February 2012, when he called Israel a "cancerous tumour" and vowed to support anyone confronting it.
He also used that opportunity to warn against any US strikes on Iran over its nuclear program, saying the US would be damaged "10 times over."
Friday's sermon came amid high tensions between the US and the Middle East.
After General Soleimani was killed the strike in Baghdad, Iran launched a barrage of ballistic missiles targeting US troops in Iraq .
As Iran's Revolutionary Guard braced for an American counter attack that never came, it mistakenly shot down a Ukrainian jetliner shortly after it took off from Tehran's international airport, killing all 176 passengers on board, mostly Iranians.
Ayatollah Khamenei called the shootdown of the plane a "bitter accident" that saddened Iran as much as it made its enemies happy.
Authorities concealed their role in the tragedy for three days, initially blaming the crash on a technical problem. Their admission of responsibility triggered days of street protests, which security forces dispersed with live ammunition and tear gas.
Ayatollah Khamenei has held the country's top office since 1989 and has the final say on all major decisions. The 80-year-old leader openly wept at the funeral of General Soleimani and vowed "harsh retaliation" against the United States
Tensions between Iran and the United States have steadily escalated since Mr Trump withdrew the US from Iran's 2015 nuclear deal with world powers, which had imposed restrictions on its nuclear program in exchange for the lifting of international sanctions.
The White House has since imposed crippling sanctions on Iran, including its vital oil and gas industry, pushing the country into an economic crisis that has ignited several waves of sporadic, leaderless protests.
Mr Trump has openly encouraged the protesters - even tweeting in Farsi - hoping that the protests and the sanctions will bring about fundamental change in a longtime adversary.
After General Soleimani was killed, Iran announced it would no longer be bound by the limitations in the nuclear agreement.
European countries who have been trying to salvage the deal responded earlier this week by invoking a dispute mechanism that is aimed at bringing Iran back into compliance and could result in even more sanctions.
Ayatollah Khamenei was always sceptical of the nuclear agreement, arguing that the United States could not be trusted.
But he allowed President Hassan Rouhani, a relative moderate, to conclude the agreement with President Barack Obama. Since Mr Trump's withdrawal, he has said there can be no negotiations with the United States.