The three-minute internet video, which was posted on Thursday on a website often used by pro-al-Qaeda groups, showed Osama bin Laden's right-hand man speaking directly to the camera with an automatic rifle propped up behind him.
There was no indication when the video was made, but its posting coincides with some of the deadliest fighting in Afghanistan since US-led forces toppled the Taliban government in 2001, with about 1,000 violent deaths this year, including 47 American troops and 18 other foreign soldiers.
Al-Zawahiri said: "Muslim brothers in Afghanistan, and especially in Kabul, stand as one with the mujahidin (Muslim fighters) so that the invading forces might be expelled.
"Do not trust these infidel invaders or their agents who want to transform you into oppressed, enslaved people."
But Afghanistan's president called al-Zawahiri "the enemy of the Afghan people" and blamed him for his country's massive suffering.
"He is first the enemy of the Afghan people, and then the enemy of the rest of the world," said Hamid Karzai on Thursday.
"He killed Afghans for years, thousands, and then he went to America and destroyed the twin towers," Karzai said. "We in Afghanistan want him arrested and put before justice."
Karzai said the Egyptian-born al-Zawahiri "is the one who destroyed our mosques and schools, vineyards and orchards."
An international force led by Nato is preparing to move into the southern heartland of the Taliban, which will allow the United States to pull out about 3,000 of the 23,000 troops it has in Afghanistan.
In the video, al-Zawahiri cited unspecified US "aggressions" in Afghan cities as examples of foreign "crimes against Islam" and mentioned what he called the "ridiculing of our holy prophet by the Italian, Danish and French", referring to the caricatures of the Prophet Muhammad which caused an uproar earlier this year.
Al-Zawahiri last appeared on a video aired by Aljazeera on June 9, in which he urged Palestinians to boycott a referendum on a statehood proposal that implicitly recognised Israel.