Al-Qaeda's number two has called US President George W Bush a "liar" who is losing his war against the network.
Zawahri has previously warned the US of interfering in the Middle East
In a video published on the Internet, Ayman al-Zawahiri called Mr Bush a "lying failure" and said al-Qaeda was stronger than ever.
The message follows a video issued for the anniversary of 9/11, in which Zawahiri said Western forces were doomed in Iraq and Afghanistan.
In the new message, he also spoke about Darfur and attacked Pope Benedict XVI.
The Egyptian militant, who is seen as the group's ideologue, has eluded capture despite a $25m bounty on his head.
In the latest video, Zawahiri said: "We have gained more strength and we are more insistent on martyrdom.
"Bush, oh failure and liar, why don't you be courageous for once and confront your people and tell them the truth about your losses in Iraq and Afghanistan."
He urged Muslims to fight a holy war in the troubled Sudanese region of Darfur against "crusaders" masked as United Nations troops.
He also called Pope Benedict XVI a "charlatan" because of his remarks on Islam, Reuters reported.
"This charlatan accused Islam of being incompatible with rationality while forgetting that his own Christianity is unacceptable to a sensible mind," he said.
The Pope caused controversy earlier this month when he quoted a medieval text which said Prophet Muhammad had brought the world only evil.
He expressed regret following angry reactions from throughout the Muslim world to the words in a speech made in southern Germany.