It comes amid tensions between the US and Iran over the possible revival of the 2015 nuclear deal.
The bombers took off from the Royal Air Force base at Fairford, England, and flew over the eastern Mediterranean, the Arabian Peninsula and the Red Sea on Sunday.
The planes took part in training missions with Kuwaiti and Saudi warplanes before departing the region.
“Threats to the US and our partners will not go unanswered,” Lt Gen Alexus Grynkewich, the top US Air Force officer in the Middle East said.
“Missions like this showcase our ability to combine forces to deter and, if necessary, defeat our adversaries.”
On Monday, Iran's semi-official Tasnim news agency said the Iranian Navy had taken delivery of several new military vessels and a submarine.
The agency unveiled the country's new stealth combat patrol boat, the Shahid Soleimani, in a post on Twitter.
The US military’s Central Command did not mention Iran in its statement about the flyover.
But Washington has previously dispatched B-52 bombers to the region as hostilities simmered between the US and Iran, the last coming in June.
Former president Donald Trump’s decision to withdraw the US from Iran's landmark nuclear deal with world powers sparked a series of escalating incidents in the region.
Iran’s navy seized two US sea drones in the Red Sea last week, only days after the country’s paramilitary Revolutionary Guard towed another sea drone before releasing it as a US warship trailed it.
The US Navy has been deploying ultra-endurance aerial surveillance drones to monitor threats in the crucial waterways, which have seen a series of maritime attacks.
Tensions also remain high after recent confrontations between US forces and Iranian-backed militias in the region. Washington last month carried out air strikes in eastern Syria that attacked areas used by militias backed by Iran’s Revolutionary Guard, prompting a response from Iranian-backed fighters.
US and Iranian negotiators in Vienna have been attempting to revive the 2015 nuclear deal, which imposed sharp limits on Iran’s atomic programme in exchange for international sanctions relief.
Last week, the State Department described Iran’s latest negotiating position as “not constructive.”
Meanwhile, Iran now enriches uranium up to 60 per cent purity — a level it never reached before that is a short, technical step away from 90 per cent.
While Iran long has maintained its programme is peaceful, non-proliferation experts warn Tehran has enough 60 per cent-enriched uranium to reprocess into fuel for at least one nuclear bomb.