In a short audio broadcast on loyalist satellite TV channels, the fugitive leader called on all of Libya's tribes to rally and expel what he called foreign agents from the country.
"Libya is for the Libyan people and not for the agents, not for imperialism, not for France, not for Sarkozy, not for Italy," he said. "Tripoli is for you, not for those who rely on Nato."
Rebels, backed by Nato air power, overran Tripoli this week and burst into Gaddafi's fortified compound, which they ransacked without finding him or his family.
A hint of desperation had crept into the maverick leader's speeches in recent weeks as successive rebel victories suggested the tide was turning against him after more than five months of civil war.
But speaking from an unknown location, he sounded more upbeat in Thursday's broadcast, suggesting the loss of most of his capital and moves by several Arab states to put their full weight behind the rebel leadership have done little to dampen his spirits.
"The enemy is delusional, Nato is retreating. It cannot go on forever in the air. Nato be damned," he shouted. "We will defeat them with determination, through will, commitment to freedom, sovereignty, dignity and glory. Never be afraid of them, only fear God, you are closer to God than they."
In a nod to a speech he made early in the uprising against his 42-year rule, Gaddafi urged Libya's youth and tribal leaders to take control of their neighbourhoods from the rebels.
"Street by street, alleyway by alleyway, house by house," he cried. "The tribes that are outside of Tripoli must march on Tripoli. Each tribe must control its area and stop the enemy setting its foot on this pure land."
"O sheikhs of the mosques, O scholars, incite the people to jihad. Go out as their leaders."
"Do not leave Tripoli to those rats, kill them, defeat them quickly. You are the crushing majority... There will be no safe place for the enemies."
He said Libyans must show the rebels no mercy.
"I call upon you to organise a million man march that will fight this time, that will fill the streets and fill the squares. Do not fear the shelling. These are blank shells that scare you."
"Bring out the men and women this time to purify Tripoli. Bring out the young inside Tripoli to protect all alleyways and districts... They must fight and not give up."
The new Libyan leadership, the National Transitional Council based in the eastern city of Benghazi, has offered a reward for Gaddafi's capture and the International Criminal Court has charged him with crimes against humanity.
