The 14-minute audio message, believed to be from al-Zawahiri, was put online Friday, CNN reported.
"The crusaders, the seculars, the Americanized army, [former President Hosni] Mubarak's thugs and some members of Islamic parties with the support of Gulf money and American plotting, all agreed to topple Mohamed Morsi's government," the message said.
Those behind Morsi's ouster want a "secular, pro-American president to rule Egypt so they can continue with their plotting -- along with the Americans and Zionists -- to divide Egypt, just like what happened in Sudan," the message said.
The message denounced U.S. treatment of hunger-striking detainees at its Guantanamo Bay prison.
"Every Muslim in every spot on Earth must work to defend the blood of Muslims that is being shed by America and its allies, and their sanctities that they are violating, and their villages and homes that they are destroying, and their wealth that they are stealing," the message said.