In an audio message broadcast on state TV late on Wednesday, he described those who carried out the bombardment as "criminals" and "barbarians".
Nato has said it regrets any civilian deaths, but that the targeted residence was a "command and control centre".
Italy's foreign minister earlier called for an immediate halt to hostilities.
Francisco Frattini said a ceasefire was necessary to allow humanitarian aid to be delivered to the war-torn country.
He also urged Nato to provide more details of its aerial campaign and precise guidelines on "dramatic errors involving civilians".
But Nato's Secretary General, Anders Fogh Rasmussen, said the alliance's air campaign was protecting civilians and would continue.
He repeated that Nato was investigating Libyan reports that seven people had been killed in an air strike last week, but stressed that it was Col Gaddafi's forces, not Nato, who were targeting civilians.
'Back to the wall'BBC Middle East editor Jeremy Bowen, who is in Tripoli, says Col Gaddafi was as defiant as ever in his latest audio message.
He said Libyans would fight "foreign barbarians" and "crusaders" to the death.
Nato's secretary general said its air campaign was protecting civilians and would continue The devil, he added, would be ashamed of the lies and allegations being made by Libya's enemies.
Col Gaddafi said he had his "back to the wall", but that the battle would "continue to the beyond, until you are wiped out".
Our correspondent says Col Gaddafi dwelt on the Nato air strike that destroyed the house of his close ally and adviser, al-Khuwailidi al-Humaidi, in Sorman on Monday.
Among the dead, who were buried a few hours before the speech, were three of Mr Humaidi's grandchildren and his daughter-in-law.
Nato said the attack was on a legitimate military target, as the house was a command centre. The Libyan government rejected the allegation.
Col Gaddafi referred to Nato's statement that its surveillance had provided conclusive proof. If the intelligence had been so good, he said, surely Nato would have known that children were living there.
"You said: 'We hit our targets with precision.' You murderers!" he said. "One day we will respond to you likewise and your homes, sons and children may become one day legitimate targets."
He called on the UN Security Council other than "the three criminals" - the US, UK and France - to send a mission to investigate the deaths of civilians in air strikes.
Nato's mission - to enforce a no-fly zone over Libya to protect civilians using "all necessary measures" short of a ground invasion - began in March in response to Col Gaddafi's violent response to an uprising.
The intervention was mandated by the UN Security Council, and led by France, Britain and the US until the end of March, when Nato took over.
Having initially been given 90 days - which would have run out on 27 June - the mission has been extended for a further 90 days.
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