The US Central Command (Centcom) said four US service members were medically evacuated to Jordan hospitals but have since been discharged. Others who had minor injuries have returned to duty.
US military officials did not disclose the identities of those killed, nor provide details about the circumstances or location of the incident. BBC reported.
It followed a week of renewed hostilities in which Washington reimposed its blockade of Iranian ports and Tehran declared the Strait of Hormuz closed, as their preliminary ceasefire collapsed less than a month after it began.
Late on Saturday, Iran's Supreme Leader Mojtaba Khamenei said in a written statement that America's "repeated breaches" of the agreement had "laid bare a fundamental truth: the signature of the US president is utterly worthless and devoid of credibility".
Khamenei, who has not been seen in public since the attack which killed his father at the start of the war, accused the US of seeking to "escalate the conflict" and said Iran had "unforgettable lessons in store for it".
Jordan's military earlier said it had intercepted 10 Iranian missiles fired into its airspace overnight, without reporting any damage.
The US death toll in the conflict has now risen to 16 after an American Navy pilot who went missing earlier this month was declared dead, marking the second increase in the toll this week.
In a statement on Saturday, Centcom said: "Two US service members in Jordan were killed in action as US Central Command (Centcom) and partner forces defended against Iranian ballistic missile and drone attacks. Additionally, one service member is currently missing.
