Syria has accused the US of carrying out a "brutal massacre" with a bombing attack in Deir al-Zour province.
The overnight strikes killed an estimated 100 pro-government fighters near the Euphrates river.
The Syrian Ministry of Foreign Affairs said it had written to the United Nations, demanding international condemnation.
The US said it was responding to an "unprovoked attack" on allied Kurdish and Arab fighters.
Syria's foreign ministry described it as "a war crime and a crime against humanity" and said the US was directly supporting terrorism.
The Middle Euphrates Valley serves as an informal demarcation line in eastern Syria, with the government controlling the western side and the US-backed SDF the east.
The two sides have clashed there in the past year, while trying to drive Islamic State (IS) militants from their last major stronghold in the country.
The Syrian pro-government forces that were hit had allegedly tried to take ground east of the River Euphrates, captured from IS by the SDF.
Elsewhere in Syria on Thursday, government warplanes bombed towns in the rebel-held Eastern Ghouta region, near the capital, Damascus, for a fourth day.
The Syrian Observatory for Human Rights, a UK-based monitoring group, said at least 36 civilians were killed, bringing the death toll to 185 since Monday.
The Syrian government has also been accused of using chemical weapons on a rebel-held town in Idlib province earlier this week.
What happened in Deir al-Zour?
The US-led coalition against IS accused pro-government forces of initiating "an unprovoked attack against well-established SDF headquarters" late on Wednesday.
"Coalition service members in an advise, assist, and accompany capacity were co-located with SDF partners during the attack 8km east of the agreed-upon Euphrates river de-confliction line," a statement said.
"In defence of coalition and partner forces, the coalition conducted strikes against attacking forces to repel the act of aggression," it added, asserting its "non-negotiable right to act in self-defence".
Unnamed US military officials subsequently told reporters that about 500 pro-government fighters, backed by artillery, tanks, multiple-launch rocket systems and mortars, were involved in the assault.
One SDF fighter was reportedly wounded in the incident. There were no American casualties.
The Syrian Observatory for Human Rights confirmed there had been an attack on SDF positions near the town of Khusham, 10km south-east of Deir al-Zour city, and put the death toll at about 20.
Why did the pro-government forces attack?
"We suspect Syrian pro-regime forces were attempting to seize terrain SDF had liberated from [IS] in September 2017," one US official told Reuters news agency.
The forces were "likely seeking to seize oilfields in Khusham that had been a major source of revenue for [IS] from 2014 to 2017, the official said.
Before the war, the Omar oil field was producing 30,000 barrels of oil per day, while the Conoco gas field was producing 13m cubic metres of gas per day.
The US-led coalition had observed a slow build-up of pro-government forces in the area over the past week and had alerted Russia, which backs the Syrian government, to the presence of SDF forces in the area, according to the official.
What does Syrian media say?
State media reported that the US-led coalition had bombed "popular forces" fighting IS and SDF forces east of the River Euphrates, denouncing what they called a "new aggression".
An Al-Ikhbariyah TV correspondent said the bombing left "dozens of dead and wounded" and identified the pro-government fighters as "local people".
The Syrian Observatory said they were local tribesmen loyal to President Bashar al-Assad and Afghan Shia militiamen.
What does Russia say?
The Russian Defence Ministry said the Syrian militia were carrying out an anti-IS operation, which was not co-ordinated with Russia, when they were attacked by the US-led air strike.
In a statement, it said 25 militia were wounded, and accused the US of being motivated by economic concerns as the strike took place near an oil refinery.
The incident "again showed that the US is maintaining its illegal presence in Syria not to fight the Islamic State group, but to seize and hold Syrian economic assets", it said.