US President Donald Trump abandoned a landmark 2015 nuclear deal between major powers and Tehran in May and has since reimposed crippling unilateral sanctions.
“What the United States wants of (the Middle East) today is enslavement,” Rouhani told an Islamic unity conference in Tehran.
Instead of “rolling out the red carpet for criminals,” Muslim governments should unite against the United States and “the region’s cancerous tumour”, Israel, he said.
Israeli Prime Minister Benjamin Netanyahu is a staunch Trump ally and a bitter critic of the 2015 deal.
Rouhani urged Iran’s rival Saudi Arabia to end its dependence on “insulting” US military aid.
“We are ready to defend the Saudi Arabian people’s interests against terrorism and superpowers with all our might,” he said.
“We do not ask $450 billion for it and will not insult you.”
Riyadh and Tehran have supported opposing sides in devastating civil wars in Syria and Yemen.