The armed men ordered drivers out of their vehicles and opened fire on the buses - setting fire to fuel tanks of at least 20 buses, according to reports.
Thousands of people were to leave the last rebel-held parts of the northern city of Aleppo in exchange for residents leaving Fuaa and Kafraya two Shia enclaves in rebel-held Idlib province.
It has left up to 50,000 civilians trapped in rebel-held East Aleppo and thousands more in the two regime towns, where the truce agreement has been repeatedly broken over the weekend.
An unknown group called Saraya al-Tawheed claimed responsibility for the attack, a jihadi-group which announced its formation yesterday.
It is claimed that these were jihadi-aligned fighters opposed to the population transfer agreed between the opposition and regime.
"The group only appeared yesterday. If it's real, it's likely, as its statement suggests, composed of people from a variety of factions who reject the deal," said Aymenn al-Tamimi, a researcher on Syrian militias.
Fateh al-Sham Front, which was formerly known as the al-Nusra Front before renouncing ties with al-Qaeda, disagreed with Ahrar al-Sham over the deal, the Syrian Observatory for Human Rights have said.
The analyst said Soraya al-Tawheed could be made up of break away fighters from this group or the jihadi organisation Jund al-Aqsa.
Syrian state media blamed "armed terrorists" for burning and destroying five buses. Damascus and pro-regime media refer to all rebel groups as "terrorists".
The Free Syrian Army issued a statement shortly after the attack condemning the "individual" and "reckless" act.
Activists have accused the Syrian regime of carrying out the attack on the buses in another way of hampering the evacuation deal.
Video allegedly of the attackers showed fighters who did not appear to be jihadi-aligned from their appearance.
State media claimed that despite the attack, buses carrying people from Aleppo have started to leave.
However, witnesses said that regime forces had blocked the evacuation route and didn't allowed the convoy to leave Aleppo because of buses burning in Idlib.
"There's collective will for the deal to stay in place. There must be solutions for all obstacles," a military source told AFP.
Dozens of buses on Sunday began entering the last rebel-held parts of east Aleppo to resume the evacuation of thousands of increasingly desperate trapped civilians and rebels.
The buses started entering the remaining rebel-held neighbourhoods on Sunday under the supervision of the Red Crescent and the International Committee of the Red Cross "to bring the remaining terrorists and their families out", state news agency SANA said.
The operation was suspended on Friday leaving up to 50,000 people trapped inside the rebel enclave without access to food or shelter, following the earlier evacuation of thousands more under a deal allowing the regime to take full control of the city.
The main obstacle to the resumption of the operation had been a disagreement over the number of people to be evacuated in parallel from Fuaa and Kafraya.
A rebel representative told AFP on Sunday that a new agreement had been reached under which the evacuations would take place in two phases.
"In a first step, half of the people besieged in Aleppo will leave, in parallel with the evacuation of 1,250 people from Fuaa," representative said on condition of anonymity.
According to the United Nations, around 40,000 civilians and rebels are trapped in the opposition-held sector of Aleppo.
"In a second step, 1,250 people from Kafraya will leave in parallel with the evacuation of the remaining people in Aleppo," the rebel representative said.
Another 1,500 people will later leave Fuaa and Kafraya along with the same number from Zabadani and Madaya, two rebel towns besieged by the regime in Damascus province.
Aleppo has seen some of the worst violence of the nearly six-year war that has killed more than 400,000 people.
Since 2012, Assad's regime has launched numerous bids to recapture opposition-held areas of Aleppo, resorting to near-daily airstrikes and barrel bomb attacks despite UN criticism.
Then last month, Russian-backed regime forces stepped up the offensive in a bid to crush the rebellion there once and for all.
The UN Security Council is expected on Sunday to vote on a French-drafted resolution demanding immediate and unconditional access for the UN and its partners to besieged parts of Aleppo and throughout Syria to ensure the delivery of humanitarian aid.