Syria has accused the US of carrying out a "brutal massacre" with a bombing attack in Deir al-Zour province.
The overnight air strikes killed an estimated 100 pro-government fighters near the Euphrates river, according to the US.
The Syrian foreign ministry said it had written to the United Nations, demanding international condemnation.
The US claimed a right to self-defence, saying it was responding to an attack on allied Kurdish and Arab fighters.
It happened in the Middle Euphrates Valley, which serves as an informal demarcation line in eastern Syria. The government controls the western side and the US-backed Syrian Democratic Forces (SDF) the east.
The two sides have clashed over the past year while trying to drive Islamic State (IS) militants from their last major stronghold in the country.
Syria described the latest strikes as "a war crime and a crime against humanity", and said the US was directly supporting terrorism.
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.
Were there Russian casualties?
A Pentagon official said Russian mercenaries were among the dead after the strike, US media report. If confirmed, this would be the first time US forces have killed Russians in Syria.
However, the Russian Defence Ministry said it had no service personnel in the area. It said it was aware of 25 Syrian militia, who had been wounded in the strikes, but no casualties.
Russia accused the US of being motivated by economic concerns, as the strikes took place near an oil field.
Pentagon officials also said they believed Russia was trying to seize control of local oil operations.
Where did the US strikes happen and why?
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.
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 (6 miles) south-east of Deir al-Zour city, and put the death toll at about 20.
What should happen to IS fighters in Syria and Iraq?
The rapid rise and fall of 'Islamic State'
The war against ‘Islamic State’ in maps and charts
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 added.
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.
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