The US-led coalition commander first attempted to get the pro-Assad forces out of the zone by dispatching two US aircraft as "a show of force".
U.S. warplanes have struck Syrian pro-regime forces in a mission to protect British and American soldiers in the country's southeast.
There were no immediate details on any civilian injuries.
The US has been backing the Maghawir and Jaysh al-Thawra fighters in the near the Syria, Jordan, Iraq border.
USA military officials have not yet determined if Syrian army forces were targeted in the strike or if they were militias aligned with the government of Syrian President Bashar Assad.
A bulldozer and a tank were hit, but US officials said it was unclear on Thursday whether they had struck the Syrian army or just militias allied with the government.
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At the time of the strike, pro-Assad forces "were advancing well inside" the de-escalation zone, the coalition said in a Thursday statement.
A day earlier, the US -led coalition struck Syrian forces near Tanf, as the forces were advancing toward an area controlled by the USA -led rebels, where British and USA bases are reportedly located in Tanf.
United States defence secretary Jim Mattis denied the destruction of the convoy, which included a number of tanks, marked an escalation in the American role in Syria.
Syrian state media gave no precise toll in the attack, but the Syrian Observatory for Human Rights, a Britain-based monitor, reported eight killed, with "most of them non-Syrian".
The U.S. -led coalition is battling the Islamic State, also known as ISIS, in Syria and is generally not attacking Assad. There were USA advisers operating at the base at the time of the airstrikes.
Syria also condemned the strike as a "brazen attack" and said it would "not be intimidated" after the surprise assault.
Tensions in the area have escalated as Syrian government forces and USA -backed Syrian forces continue to advance closer to each other.
"Such actions that were carried out against the Syrian armed forces. this is completely unacceptable". Last week, BuzzFeed reported that USA special forces have been increasingly fighting alongside these rebel groups in Southern Syria, a region that has seen an increasing amount of ISIS activity as the group has lost territory elsewhere.