On the tape, he called the US President, George W Bush, a “butcher” and a “failure.”
The targeted attack on a village near Peshawar killed 18 civilians, including women and children.
Pakistani officials claim as many as five of them were al-Qaeda militants.
“They said this was intended to kill myself and four brothers but now the whole world has discovered their lies,” said al-Zawahiri.
“The whole world has discovered the magnitude of the United States’ lies and failure and how barbaric it is in its fight against Islam and Muslims,” he said.
“Butcher of Washington, you are not only defeated and a liar, but also a failure. You are a curse on your own nation,” and asked, “Bush, do you know where I am? I am among the Muslim masses.
Al-Zawahiri also referred to an olive branch by Osama bin Laden in a tape on January 19 in which the al-Qaeda leader offered the US an “honourable exit” from Iraq.
He warned that the refusal to accept the offer meant that Americans could expect “a future coloured by blood, the smoke of explosions and the shadows of terror.”
It is the first appearance by al-Zawahiri since the missile strike on January 13.
In an earlier appearance on January 6, he called on President Bush to admit defeat in Iraq and Afghanistan.