The move was aimed at protecting American forces from attacks by the Tehran-backed groups, the US Central Command said in a statement.
Iranian foreign ministry spokesman Nasser Kanani denounced the strikes, calling them a "violation of the sovereignty, independence and territorial integrity" of Syria.
In a televised statement, Mr Kanani also called on US troops to leave Syria, denying any Iranian involvement in the support of fighters on the ground.
"The sites targeted had no links to the Islamic Republic," Mr Kanaani said.
The US strikes came in response to an August 15 attack on a compound used by coalition and US-backed Syrian opposition fighters.
US President Joe Biden gave the order for the attack, said army spokesman Col Joe Buccino.
The US Central Command called the strikes a “proportionate, deliberate action intended to limit the risk of escalation and minimise the risk of casualties”. It did not offer information of casualties.
“Today’s strikes were necessary to protect and defend US personnel,” said Col Buccino.
“The United States does not seek conflict but will continue to take necessary measures to protect and defend our people. US forces remain in Syria to ensure the enduring defeat of ISIS.”
It is not the first time US warplanes have struck Iran-backed forces in Iraq and Syria. The US hit operational and weapons storage facilities at two sites in Syria and one in Iraq last June.
US forces were posted to Syria during the Obama administration's campaign against ISIS, teaming up with the Kurdish-led Syrian Democratic Forces. There are about 900 US troops in Syria, most of them in the east.
But Iran-backed militias established a foothold in Syria while fighting in support of President Bashar Al Assad during Syria's civil war.
Such militias are heavily concentrated west of the Euphrates in Deir Al Zor, where they receive supplies from Iraq through the Al Bukamal border crossing.
Drones launched by Iranian-backed militias
On August 15, drones allegedly launched by Iranian-backed militias struck Al Tanf Garrison, which is used by US forces.
US Central Command at the time described the assault as causing “zero casualties and no damage”.
Deir Al Zor is a strategic province that borders Iraq and contains oilfields. Iran-backed militia groups and Syrian forces control the area and have often been the target of Israeli war planes in previous strikes.
The strikes hit when Syrian Foreign Minister Faysal Mikdad was visiting Moscow.
Mr Mikdad called for the withdrawal of US and Turkish occupation forces from Syria, state news agency Sana reported on Tuesday.
In a joint press conference with Russian counterpart Sergey Lavrov, he said: “We appreciate Russia’s support for Syria in its war against western-backed terrorism.
“The US state is the side which supports terrorism in Syria, destroys its infrastructure and economy, and continues to plunder its resources and impose coercive economic measures in flagrant violation of [the] UN charter.”
Hundreds of US troops are stationed in Syria's north-east as part of a coalition focused on fighting remnants of ISIS.
There was no immediate confirmation of the US strikes from Syrian state media.
Revolutionary Guard general killed
Earlier, Iranian state media said a Revolutionary Guard general "on a mission in Syria as a military adviser" had been killed on Sunday.
The reports did not say how he was killed, only describing him as a "defender of the sanctuary", a term used for those who work on behalf of Iran in Syria or Iraq.
Iran says it has deployed forces in Syria at the invitation of Damascus and only as advisers.
The Britain-based Syrian Observatory for Human Rights on Wednesday said the strikes killed six fighters who were guarding the targeted facilities.
The monitor, which relies on a wide network of sources in Syria, said those killed were Syrians and non-Syrians.
It said Iran-backed Afghan Shiite fighters were among the groups operating in the region.
Arms depots and a training camp for Iran-backed militia fighters were among the targets hit, the monitor said.