The "proportionate" strikes targeted "Kataib Hezbollah militia group and other Iran-affiliated groups", Defence Secretary Lloyd Austin said.
He said the precision strikes were "in direct response" to attacks against US and coalition allies in Iraq and Syria.
A number of US military personnel were injured in a missile attack on an airbase in western Iraq last week.
The US military's Central Command (CentCom) said at the time that an Iran-backed militia targeted the Al Asad airbase, which hosts American troops, with ballistic missiles and rockets.
A group calling itself the Islamic Resistance in Iraq claimed responsibility for that attack.
According to the US-based Washington Institute for Near East Policy, the group emerged in late 2023 and is comprised of several Iran-affiliated armed groups operating in Iraq. It has claimed other attacks against US forces in recent weeks.
In a statement, Mr Austin praised the "professionalism" of US military personnel in planning and conducting Wednesday's strikes in Iraq as part of efforts "to further dismantle and degrade ISIS [the Islamic State group]."
Mr Austin stressed that he and US President Joe Biden "will not hesitate to take necessary action" to defend American interests.
"We do not seek to escalate conflict in the region. We are fully prepared to take further measures to protect our people and our facilities. We call on these groups and their Iranian sponsors to immediately cease these attacks," he said.
In a separate statement, CentCom said Wednesday's strikes in Iraq were carried out at 00:15 local time (21:15 GMT Tuesday) in response to the attack - among others - on Al Asad airbase in western Iraq on 20 January.
"These strikes targeted [Kataib Hezbollah] headquarters, storage, and training locations for rocket, missile, and one-way attack UAV capabilities," CentCom said.
The US military provided no details on where the targeted militia facilities were located in Iraq.
Saturday's attack on Al Asad airbase followed a US drone strike in Baghdad earlier this month in which a pro-Iran militia leader was killed.
US troops in Iraq and Syria have been attacked dozens of times by Iran-aligned militants since the Israel-Gaza war started in October.
The US military and its allies - including the UK - have also intervened to stop Houthi missile attacks on international shipping in the Red Sea.
Washington and London have carried out strikes against the Houthis - an Iran-backed rebel group controlling much of western Yemen.
The Iranian military has carried out a number of missile strikes over the last week against targets in Syria, Iraq and Pakistan.
Last Saturday, Iran accused Israel of carrying out an airstrike in the Syrian capital Damascus, which killed five senior members of Iran's security forces.
bbc