Despite heightened tensions between the longtime foes, Washington has offered humanitarian assistance to Iran while it struggles with the coronavirus outbreak.
Iran is the most-affected country in the Middle East with over 1,500 coronavirus deaths and 20,610 infected people.
"Several times Americans have offered to help Iran to contain the virus ... You are accused of creating this virus. I do not know whether it is true, but it is strange that you want to help Iran," Khamenei said.
"Aside from the fact that you have shortages in your fight against the virus, what if you give us a drug that will help the virus to remain in Iran permanently?"
Khamenei, an anti-U.S. hardliner, said the Islamic Republic had the capability to overcome "any kind of crisis and challenges, including the coronavirus outbreak."
Tensions have been running high between Iran and the United States since 2018, when U.S. President Donald Trump exited Tehran's 2015 nuclear deal with six world powers and reimposed sanctions that have crippled the country's economy.
Frictions increased when Trump ordered a U.S. drone strike that killed top Iranian military commander Qassem Soleimani on Jan. 3.. Iran retaliated by hitting US targets in Iraq on Jan. 8.
"Our number one enemy is America. It is the most wicked, sinister enemy of Iran ... its leaders are charlatans," Khamenei said. Iranian authorities have blamed US sanctions for hampering its efforts to curb the outbreak and called for the restrictions to be lifted. Washington has refused to lift sanctions.