Essam al-Rayes' comments come after tensions between local rebel groups and Al-Nusra over the capture of a border crossing between Syria and Jordan.
"We reject all forms of cooperation with Al-Nusra Front because keeping silent on its excesses, its statements and its violations will only allow them to continue," Rayes told AFP.
Speaking via Skype, he said the groups fighting under the Southern Front banner hoped their position would "tell Syrians that Al-Nusra's ties with Al-Qaeda push the revolution away from its path and objectives."
"We do not want Syria to become a base for jihad, or the expansion of the Islamic State (IS)," he added.
The comments came after several similar individual statements from battalions fighting in southern Syria.
Ties between the groups and Al-Nusra have been strained since the April 2 takeover of the Nasib border crossing.
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The Al-Qaeda affiliate participated only in the last stages of the battle for the crossing, playing no role in the planning or initial attack.
Despite that, Al-Nusra fighters seized parts of the border post, creating considerable resentment among the groups that carried out the operation.
They asked Al-Nusra to withdraw from the border point and the duty-free zone between the two crossings, which it did last week.
In recent days, the rebels have publicly disassociated themselves from the group.
Despite his comments, Rayes said the groups had no intention of fighting against Al-Nusra.
"This is not a declaration of war; we don't want any tensions on the ground because our battle is not against Al-Nusra."
Syrian rebel forces remain prominent in the south, unlike in the north, where Al-Nusra and the rival IS have seized large swathes of territory.