"O nation of jihad, support your sons so that we defeat our enemies and rid our homeland of their slaves," the recording said.
Gadhafi is called "an enemy of Islam."
The Fighting Islamic Group was formed in the 1990s with Libyan veterans of fighting against the Soviet army in Afghanistan as its base. A man identified as Abu Laith al-Libi, described on the tape as the group's leader, promises its members will become "the faithful soldiers of Osama bin Laden."
Zawahiri, now believed to be in Pakistan with bin Laden, is a native of Egypt.