Al-Zawahiri has called for more protests outside American embassies.
Like in other messages released by al-Qaeda's Yemeni and North African branches last month, Zawahiri praised last month's assault on the US consulate in Benghazi that killed four diplomats but stopped short of claiming responsibility.
In the recording, posted on Islamist websites on Friday, he called on "free and distinguished zealots for Islam" who attacked the consulate and protested outside other American embassies to "continue their opposition to American crusader Zionist aggression against Islam and Muslims".
The recording appeared on the Mujahedin al-Ansar website which carries statements from al-Qaeda leaders.
Zawahiri said US authorities "permitted the film in the name of personal freedom and freedom of expression" but failed to practise those values in its treatment of Muslims imprisoned in Guantanamo Bay, Iraq and Afghanistan.
US President Barack Obama's administration condemned the short film, made privately and amateurishly in California, as reprehensible but said it could not be banned without violating the US constitutional right to free speech.
The Guantanamo detention camp was set up after the September 11, 2001 attacks to hold non-American captives suspected of involvement with al Qaeda, the Taliban or other Islamist militant groups. Of the 779 men who were held there, 167 remain.
Zawahiri, an Egyptian-born preacher who became al Qaeda's chief after the death of Osama bin Laden last year, also criticised Obama after his Democratic Party pledged at its pre-election convention to recognise Jerusalem as Israel's capital.
He said that decision and Obama's prayers at Jerusalem's Western Wall, made during a 2008 visit, showed Muslims faced a "crusader Zionist war" led by the United States.
In September al-Qaeda in the Islamic Maghreb (AQIM), the group's North African affiliate, praised the Benghazi attack and urged Muslims to kill more US envoys.
Earlier last month, al-Qaeda in the Arabian Peninsula (AQAP), its Yemeni wing, called for more protests and said the Benghazi attackers had been partly motivated by the death of an AQAP leader in a US drone strike. – Reuters