Washington had said Iran's only chance of avoiding the sanctions would be to accept Trump's offer to negotiate for a tougher nuclear deal. "Beside sanctions, they are talking about war and negotiations", he said via his official Twitter account in English.
Washington reimposed the sanctions last week after pulling out of a 2015 global deal aimed at curbing Iran's nuclear program in return for an easing of economic sanctions.
"Negotiations with the USA would definitely harm us and they are forbidden", Khamenei said, adding that the Americans had proven they could not be trusted.
While E.U. nations have pledged to try to preserve economic ties with Iran, most large European companies already concede they will sacrifice trade with Iran, rather than risk suffering from secondary sanctions.
"Negotiating the nuclear agreement was a miscalculation, and I personally made a mistake in these negotiations", Khamenei said.
With the nuclear deal crumbling, Rouhani finds himself with little to show for his five years in power and increasingly under fire from all sides. "Even if we were to negotiate with the USA, we would never hold talks with the current USA administration".
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A significant cut in oil sales is also expected when the US reimposes a second phase of sanctions in November - with some analysts estimating a drop of 700,000 barrels per day from its current level of 2.3 million.
Protests in cities and towns across Iran have often begun with slogans against the high cost of living, high prices and a lack of jobs, but have then quickly turned into anti-government rallies.
There was also a show of military resolve as Defence Minister Amir Hatami unveiled a next generation short-range ballistic missile and vowed to further boost the country's missile capabilities.
Iranian media quoted Khamenei as saying in a brief statement issued on August 11 that punishments for those accused of economic corruption should be "carried out swiftly and justly".
Trump has said Iran must also stop meddling in conflicts in Syria and Yemen, but Foreign Minister Javad Zarif struck a defiant tone on Monday, telling Qatar's al Jazeera TV: "Iran will not change its policies in the region because of USA sanctions and threats".