Coalition fighter jets on Thursday struck a convoy of militiamen advancing inside a protected "deconfliction zone" north-west of the southern town of al-Tanf, the military alliance said in a statement.
The US, which is leading an air campaign in Syria targeting groups such as the Islamic State of Iraq and the Levant (ISIL, also known as ISIS), said the convoy's advance had posed a threat to US and US-backed Syrian rebel forces in the area.
"This brazen attack by the so-called international coalition exposes the falseness of its claims to be fighting terrorism," a Syrian military source told state media on Friday, confirming that the bombing had killed "a number of people" and caused material damage.
Russia, which launched its own air campaign in September 2015 in support of Syria's President Bashar al-Assad, called the strike "a breach of Syrian sovereignty".
"Such actions that were carried out against the Syrian armed forces ... [are] completely unacceptable," Gennady Gatilov, Russia's deputy foreign minister, was quoted as saying by state-run RIA Novosti on Friday.
A member of the US-backed Syrian rebel forces told the Reuters news agency that the convoy comprised Syrian and Iranian-backed militias and was headed towards the Tanf base, where US special forces operate and train Free Syrian Army rebels.
Rami Abdel Rahman, head of the Syrian Observatory for Human Rights (SOHR), said at least eight people had been killed in the attack.
"Most of the killed belong to militias loyal to the Syrian regime and are not Syrians," he told the DPA news agency.
SOHR, a UK-based monitor tracking developments in Syria's long-running conflict via a network of contacts on the ground, also said that four military vehicles carrying pro-government forces and their allies were destroyed in the strike.
Tanf is part of a region known as the Badia, which consists of vast, sparsely populated desert territory that stretches all the way to the Jordanian and Iraqi borders and was declared a military priority by Syria's foreign minister earlier in May.
Two months of US-backed rebel advances against ISIL fighters have allowed them to secure swaths of territory in the Badia, alarming the Syrian government and its allies.
But rebel sources had warned last week that the Syrian army and Iranian-backed militia moved hundreds of troops with tanks to the town of Sabaa Biyar, which is in the Badia, and is near the strategic Damascus-Baghdad highway.
That highway was once a major weapons supply route for Iranian weapons into Syria.
A Western intelligence official, speaking on condition of anonymity, told Reuters that Thursday's strike sent a strong message to Iranian-backed militias that have been spearheading the advance that they would not be allowed to reach the Iraq border from Syria.
The US-led coalition did not signal it would cede ground around Tanf.
"Coalition forces have been operating in the At Tanf area for many months training and advising vetted partner forces engaged in the fight against ISIS," according to a statement by the US-led military alliance.
US officials said an agreement existed with Russia on a so-called "deconfliction" area around Tanf, meant to avoid an accidental clash of forces.
The statement by the US-led coalition acknowledged a zone but did not offer any details about it, other than to say it was still active.
"The agreed upon deconfliction zone agreement remains in effect," the statement said.
In April, the US army fired dozens of cruise missile strikes at the Syrian government-held Shayrat airbase a Syria's Shayrat airbase, in retaliation for a suspected chemical weapons attack on a rebel-held town that killed scores of civilians.
At the time, the strikes were described as a one-off measure to deter any future chemical weapons use.
Syria's civil war began in 2011 after mass protests against Assad's rule and has killed hundreds of thousands of people and driven half the country's population from their homes.
Source: Al Jazeera and news agencies