"If one of our chiefs passes, another arises in his place," al-Zawahiri said, without making a specific threat.
Al-Libi's involvement with al-Qaeda was highlighted last year by an appearance in a video with al-Zawahiri.
He was the first spokesman to announce that bin Laden had survived the US-led invasion of Afghanistan in late 2001.
According to the US media , al-Libi was believed to be behind a suicide bombing in February 2007 that killed at least 23 people outside the main Bagram base in Afghanistan during a visit by Dick Cheney, the US vice-president.
In October, the US military in Afghanistan named al-Libi among several so-called "mid-level" al-Qaeda and Taliban fighters, and offered a $200,000 reward for his capture.