One witness told the AP he saw airstrikes hit tanks and a rebel convoy, which included a passenger bus carrying fighters toward Brega.
Dramatic video made available by the rebels' National Transitional Council purported to show the moment of impact and the immediate aftermath of the airstrike.
An official at Ajdabiya hospital said at least five people were killed and 22 injured in the strike, including some with serious burns.
He said other casualties were left in the field in the chaos to flee the area.
The airstrike came as rebels forces pushed toward the outskirts of Brega, an eastern oil port that has traded hands several times since the revolt began in February.
The attack brought fresh questions about coordination between NATO and the patchwork of rebel militias.
Tensions between the rebels and NATO were flaring even before the latest accident, with the fighters criticising the alliance for doing too little to help them.
Earlier on Thursday, a rebel spokesman in Benghazi had suggested the airstrike could have been the work of Libya's government forces.
But later in the day, the rebels' top military commander described the attack as a likely NATO accident.
"We would assume that it was a NATO mistake, friendly fire," Abdel-Fatah Younes told reporters in Benghazi.
Younes, who is the chief of staff for the rebel forces, urged the alliance to provide "some answers regarding what happened today".
The commander said that if in fact the airstrike turned out to have been the work of Gadhafi's forces, it would expose holes in NATO's efforts to ground Libyan warplanes.
NATO did not directly acknowledge responsibility for a blundered airstrike on the rebels, but noted that the area where Thursday's attack occurred was "unclear and fluid with mechanised weapons travelling in all directions".
From above, both sides may appear very similar.
Rebels are using tanks and vehicles seized from the Libyan military.
The forces behind Libyan leader Moammar Gadhafi, meanwhile, are increasingly mixing into civilian areas and adopting the guerrilla-style appearance of their foes.
Last Friday, a NATO airstrike killed 13 rebel fighters in eastern Libya.
An opposition spokesman at the time described it as an "unfortunate accident" in the shifting battles and pledged support for the international air campaign to weaken Gadhafi's military power.
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