"These statements are, to say the very least, shocking and disappointing," said Nasr al-Hariri, head of the opposition's negotiations delegation, at a press conference in Istanbul.
He said De Mistura was well aware "which party has cooperated positively to advance the political process and which is the obstacle," in reference to Syria's government.
De Mistura on Tuesday urged the still-fractured Syrian opposition to form a unified delegation ahead of a next round of peace talks tentatively scheduled to take place in Geneva next month.
He added that Syria's government could not "simply announce victory" despite its recent advances, and that "the only answer is a political process".
The main opposition camp, the Saudi-backed High Negotiations Committee (HNC), whose delegation Hariri heads, insists that Syrian President Bashar al-Assad must leave office as part of any peace deal.
That stance has put the HNC at odds with two other opposition groups, known as the Moscow and Cairo platforms.
De Mistura has in recent weeks upped the pressure on the groups to unite and said he believed they would be meeting again in Riyadh in the coming weeks, after talks there last month failed to produce a compromise.
Moscow is a close ally of Syria's government and has developed another negotiations track in the Kazakh capital Astana that the opposition sees as an attempt to hijack international peace talks.
HNC chief Riad Hijab also slammed De Mistura's comments, saying on Twitter that they reflected the "defeat of UN mediation."
"Time after time, De Mistura implicates himself with unstudied statements that reinforce our call for a new UN approach on Syria," he said.
More than 330,000 people have been killed in Syria since the conflict began with anti-government protests in March 2011.
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