Zawahiri "carved a trail of murder and violence against American citizens," US President Joe Biden said as he announced the killing.
He was taken out in a CIA-orchestrated counter-terrorism operation in Kabul on Sunday.
The terrorist plotted the 9/11 attacks alongside Osama Bin Laden and took over Al-Qaeda after his former boss was killed in a 2011 US operation.
Mr Biden said: "Now justice has been delivered and this terrorist leader is no more."
US President Joe Biden announced the news in an address to the American people
POOL
Ayman al-Zawahiri took over from Osama Bin Laden in 2011
SITE Monitoring Service
He is believed to have been on the balcony of a safe house when the US drone fired two missiles at him.
Other family members were unharmed, despite being present.
US officials say Zawahiri was the only person killed in the attack.
Final approval for the "precision strike" was given by Mr Biden, who said the 71-year-old's killing had been months in the planning.
Mr Biden said: "No matter how long it takes, no matter where you hide, if you are a threat to our people, the United States will find you and take you out.
"We shall never waver from defending our nation and its people."
Ayman al-Zawahiri was on the FBI's most wanted list
FBI
As well as 9/11, Zawahiri masterminded the suicide bombing of the USS Cole naval destroyer in Aden in October 2000 which killed 17 US sailors, and the 1998 attacks on US embassies in Kenya and Tanzania, in which 223 people died.
A Taliban spokesman described the US operation as a clear violation of international principles.
They said: "Such actions are a repetition of the failed experiences of the past 20 years and are against the interests of the United States of America, Afghanistan and the region."
But Mr Biden insisted that Afghanistan would never again become a safe haven for terrorists.
A new Al-Qaeda leader will no doubt emerge but Mr Biden will hope Zawahiri's killing will bring closure to families of the nearly 3,000 victims of the 2001 attacks.