The Pentagon said the strike was ordered in response to attacks against US and coalition personnel in Iraq.
The action destroyed “multiple facilities” used by Iranian-backed Iraqi militant groups, it said.
Militia officials said one person had been killed, while a war monitor reported at least 22 fatalities.
The Pentagon said its strike near the Iraqi border in eastern Syria was a “proportionate military response” that was taken “together with diplomatic measures”, including consulting coalition partners.
It came after a civilian contractor was killed in a rocket attack on US targets earlier this month. A US service member and five other contractors were also injured when the rockets hit sites in Irbil, including a base used by the US-led coalition.
Rockets have also struck US bases in Baghdad, including the Green Zone, which houses the US embassy and other diplomatic missions.
There are about 2,500 US troops in Iraq to assist Iraqi forces in the fight against the Islamic State (IS) group.
What do we know about the air strike?
The Pentagon said the strike on Thursday was launched “at President Biden’s direction”.
It targeted facilities located at a border control point used by a number of Iran-backed militia groups, including Kataib Hezbollah and Kataib Sayyid al-Shuhada, it said.
Kataib Hezbollah and Kataib Sayyid al-Shuhada have previously carried out or supported rocket attacks targeting US assets in the country.
In its statement, the Pentagon said the operation “sends an unambiguous message”, it said in a statement..
The US did not confirm any casualties, but an Iraqi militia official told the Associated Press news agency at least one fighter was killed and a number of others wounded.
The official said the strikes hit an area along the border between the Syrian city of Boukamal and the Iraqi town of Qaim.
The Syrian Observatory for Human Rights, a UK-based monitoring group, said the US attack had killed at least 22 fighters from the Popular Mobilization Forces, an Iraqi umbrella group of mostly Shia paramilitaries that includes Kataib Hezbollah.
“The strikes destroyed three lorries carrying munitions,” the observatory’s Rami Abdul Rahman earlier told AFP. “There were many casualties.”
Kataib Hezbollah has denied any role in the recent rocket attacks targeting US personnel, but US Defence Secretary Lloyd Austin told reporters he was “confident in the target that we went after”.
“We know what we hit,” he said. “We’re confident that that target was being used by the same Shia militants that conducted the strikes” earlier this month.
Pentagon Press Secretary John Kirby said the strike was meant to punish the militias but not to escalate tensions with Iran, with whom the US is seeking to renew talks over a nuclear deal abandoned by former President Donald Trump, the New York Times reports.
Since 2009, the US has designated Kataib Hezbollah as a terrorist organisation, accusing them of threatening the peace and stability of Iraq.
Iran’s influence over Iraq’s internal affairs has grown steadily since the US-led invasion that toppled Saddam Hussein in 2003.