In an audio recording broadcast by Aljazeera on Wednesday he vowed the real battle with the United States had yet to begin.
"Rely on God and devour the Americans, like lions devour their prey. Bury them in the Iraqi graveyard," al-Zawahri said.
He also called on Palestinians to resist Israel, warning them not to lay down their weapons because "only jihad will liberate Palestine".
"We will not let America enjoy security until security becomes a fact in Palestine," al-Zawahri said, repeating what Bin Ladin had said in a previous tape.
Al-Zawahri warned al-Qaida's strikes so far were no more than "skirmishes" and the "real epic" with the United States had yet to begin in earnest.
He also exhorted Arabs to topple their rulers and urged Pakistanis to rise up against President Pervez Musharraf.
Aljazeera's videotape of al-Qaida chief Usama bin Ladin and al-Zawahri showed the pair in an "undetermined mountain area", probably recorded towards the end of April or in early May.
In the videotape, Bin Ladin praised the hijackers who crashed planes into New York's World Trade Center and the Pentagon near Washington two years ago.
"Whoever wants to be taught about loyalty and honesty should have known them… they were the most honest and the bravest," Bin Ladin said.
And he had a word of caution for those Muslims who oppose jihad.
Bin Laden and al-Zawahri appeared
in reasonable health on the tape
"I tell them that those who fear climbing up mountains will live forever in holes," he said.
The latest video could end months of speculation about Bin Ladin's fate.
In the immediate aftermath of their Afghanistan campaign, US military sources said Bin Ladin could have been killed in bombing raids.
The speculation was further fuelled after he dropped out of sight.
Meanwhile, stocks sank on Wednesday following the release of the new tape.
The tech-packed Nasdaq composite index dropped 42 points or 2.29%, to 1830.
The blue-chip Dow Jones industrial average fell 88 points or 0.93%, to 9418 while the broader Standard & Poor's 500 Index lost 11 points or 1.13%, to 1011.
"It's still a fairly orderly decline, volume is certainly not strong. I think the Bin Ladin tape is more of a rationale and an excuse to sell than anything else," said Keith Keenan, vice president of institutional trading at brokerage Wall Street Access.