Already recognised as the sole legitimate representative of the Syrian people by the Gulf states, Turkey and France, the group was snubbed by Islamist groups including the two most important, the Al-Nusra Front and Liwa al-Tawhid.
The announcement in an Internet video by the Islamist rebel groups of their decision and their aim to make Syria an Islamist state could, experts believe, hamper the coalition's pressing request for military help from the West.
"We, the fighting squads of Aleppo city and province, unanimously reject the conspiratorial project called the National Coalition and announce our consensus to establish an Islamic state" in Syria, a spokesman announced in the video.
"We reject any external coalitions or councils imposed on us at home from any party whatsoever," he said of the November 11 announcement of the new opposition umbrella grouping.
The Al-Nusra Front, a formidable fighting force, has claimed the majority of suicide bombing attacks in Syria's deadly 20-month-old conflict, while Liwa al-Tawhid, at first close to the Muslim Brotherhood, has become more radical.
"The announcement by the Islamist groups is a major blow to the coalition," said Hilal Khashan, a political science professor at the American University of Beirut.
"It is for this reason that the Americans and British remained prudent before recognising the coalition as the sole legitimate representative of the Syrian people."
But he added: "It will not have a great impact on the question of weapons because if the West really wants to aid the emergence of a liberal and nationalist coalition, it must arm the secular groups to counterbalance the Islamists."
In the Islamist video, after the statement is made another man holds up a Koran, saying forcefully to the camera: "Make this your constitution."
The video speaker listed 14 armed groups as signatories to the statement, but the Ahrar al-Sham group rejected the proclamation on its web page, saying it did not endorse the statement.
However, the Islamist groups rejecting the National Coalition are among the most important fighting units in northern Syria, which is now almost entirely in rebel hands.
Abdel Jabbar al-Okaidi, who heads the main rebel Free Syrian Army (FSA) in Aleppo, told AFP the statement did not represent the opinion of all rebel groups in the province.
"These groups represent a number of military factions on the ground and reflect their position, but not all military forces in Aleppo agree with this," the defector, a former army colonel, told AFP by phone.
"The military council has announced its support for the National Coalition and is collaborating with them."
Jihadists have become the main armed opposition fighting in northern Syria, with militants estimating their manpower could number more than 10,000.
Beirut-based scholar Francois Burgat is an expert on the Islamist movement and believes the video statement is more military than political.
"If this news is confirmed, the headlong eradication of the Syrian regime means these are the most determined fighters who are in the forefront," such as the Al-Nusra Front, he said.
"The presence of this radical fringe group does not, however, allow us to prejudge the ultimate political balance of the rebel combatants: the Front is appreciated as an armed spearhead, not as an ideological leader.
But, paradoxically, this "risks reining in those who favour lifting the arms embargo, while it is precisely such a measure that could give real credibility to the coalition and speed up the political reorientation of the armed groups," Burgat added.
Thomas Pierret, a Syria specialist at the University of Edinburgh, said he believes the West is addressing the problem the wrong way.
"The West is asking the question backwards, separating the question of representation from that of resources: they expect the Coalition to prove itself before helping it," he said.
"Yet, such organisations cannot achieve credibility unless they have resources to distribute, and such resources have to be obtained initially from abroad.
"This is a basic tenet of international politics that the West pretends not to be aware of."
The news of the video came as EU ministers meeting in Brussels explored how to assist the new coalition, including lifting the arms embargo.