The audio message, which was accompanied by a 46-page English transcript, was the first installment of answers to a raft of online questions and focused mainly on future al Qaeda efforts elsewhere in the region, particularly Egypt.
"We haven't killed the innocents, not in Baghdad nor in Morocco, nor in Algeria, nor anywhere else," he said according to the English transcript which, like the audio message, appeared on Web sites linked to the group.
The answer was in response to the question "excuse me, Mr. Zawahiri, but who is it who is killing with Your Excellency's blessing the innocents in Baghdad, Morocco and Algeria?"
Al Qaeda has taken credit for the destruction of the World Trade Center which killed nearly 3,000 people in New York City in 2001, while its affiliates in Iraq, Afghanistan and Algeria regularly set off explosives in crowded urban areas that have taken thousands of lives.
"If there is any innocent who was killed in the Mujahideen's operations, then it was either an unintentional error or out of necessity," Zawahiri added.
He went on to say that it was their opponents who killed innocents and also noted that "the enemy intentionally takes up positions in the midst of the Muslims for them to be human shields for him."
Zawahiri reassured many of the questioners, who seemed worried about the direction of the organization, that the global jihad was on track and would soon expand elsewhere.
"I expect the Jihadi influence to spread after the Americans' exit from Iraq, and to move towards Jerusalem," he said to those asking when attacks on Israel would take place.
He also predicted the end of the Saudi state, which is "swimming against the tide of history" and the government of his native Egypt, which he called a "corrupt, rotten regime (that) cannot possibly continue."
Many of the questions he chose to answer focused on restarting the jihad in Egypt, which Zawahiri himself helped begin and was crushed by the government in the 1990s.
"The days will reveal to you what you didn't know, and news will come to you from those who didn't have it," he said quoting an old Arabic proverb, about when the jihad would begin again in Egypt, and counseled patience.
Egypt's plainclothes State Security officers and uniformed police were declared "permissible to kill" in the struggle for Egypt and he hinted that he had supporters in the Egyptian army, like the man who assassinated former Egyptian President Anwar Sadat in 1981.
"The Egyptian army which produced Khalid al-Islambouli ... continues to be full of those whose hearts boil with jealousy for Islam and Muslims and who long for the opportunity to remove the corrupt gang which rules Egypt," he said.
Though the tape could not be independently verified, the message bore the logo of the Al Qaeda's media arm, al-Sahab, and appeared on Web sites linked to the organization.
Al-Zawahiri said he chose around 100 questions to answer.
Al-Sahab announced in December that al-Zawahiri would take questions from the public posted on Islamic militant Web sites and would respond "as soon as possible."
Zawahiri also addressed the issue of al Qaeda's founder, Osama bin Laden, assuring supporters that the reclusive leader was in good health.
"Sheik Osama bin Laden is healthy and well, by the grace of Allah," he said, while noting he would not be there forever. "He must die one day, whereas Allah's religion will remain."