George Sabra, interim head of the National Coalition, issued the rejection after President Bashar al-Assad's forces seized all of the Qusayr area in central Syria.
"What is happening in Syria today completely closes the doors on any discussions about international conferences and political initiatives," Sabra told a press conference in Istanbul.
He was referring to an initiative headed by Washington and Moscow to bring the regime and opposition to peace talks in Geneva.
He had already said on May 30 that the opposition would not attend a peace conference while Iran and the Lebanese Shiite movement Hezbollah were supporting troops on the ground.
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The seizure of Eastern Bweida village, the last rebel bastion in the area, brought the entire Qusayr region near the Lebanese border back under regime control.
That came four days after the town of Qusayr, which had been in insurgent hands for a year, fell to the army and fighters from the Lebanese Shiite movement Hezbollah.
But he said the opposition would refuse to be dragged in, saying that this would change "our lives in the region into hell."
Assad's regime is dominated by members of the Alawite community, an offshoot of Shiite Islam, while the rebels fighting it are mostly Sunni Muslims.
He also repeated previous statements by rebels that they have the right to defend Syria, warning the Lebanese government that it would have to take responsibility for the "implications of the invasion" by Hezbollah.
Hundreds of people who fled Qusayr had taken refuge in Eastern Bweida, 14 kilometres to the northeast, but Syrian state television broadcast footage of a desolate village devoid of signs of life.
Dozens of wounded Syrians and Lebanese from the rebel side were evacuated to Arsal, a border town in northern Lebanon, and to Baalbek in the east, security officials said.