The top general said although the United States is now focused on carrying out its economic threats against Iran, Tehran would do well to get ready to come to grips with other types of threats as well.
"You are not in a position to incite the Iranian nation against Iran's security and interests", Rouhani added, Reuters reported.
Speaking to Iranian ambassadors and senior diplomats on Saturday, Khamenei backed Rouhani's earlier warning to close the Strait of Hormuz, thus blocking all oil shipments from the Persian Gulf.
After the USA pulled out of the nuclear agreement, Iran made clear that it wants to keep the deal alive, but only if the remaining five powers can guarantee that Iran will not face economic isolation under the sanctions that the US has started to revive.
In Washington, U.S. officials familiar with the matter told Reuters that the Trump administration has launched an offensive of speeches and online communications meant to foment unrest and help pressure Iran to end its nuclear program and its support of militant groups. However, secondary U.S. sanctions remained.
"Negotiations with the Europeans do not to stop; however, we should not linger, waiting for the European pack", said Khamenei.
"The word and even the signature of the Americans can not be relied upon, so negotiations with America are of no avail", Khamenei said.
"The assumption that negotiations or establishing ties with the USA would solve the country's problems is an obvious error".
"Negotiations with the Europeans should not be stopped, but we should not be just waiting for the European package, but instead we should follow up on necessary activities inside the country (against USA sanctions)", Khamenei said.
Iranian officials, including Rouhani, have traversed the globe on a diplomatic mission to save the nuclear deal after Washington withdrew from the agreement in May.
Europe, Russia, and China all want the deal to survive.