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.
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.
Read more on BBC