Flight maps showed the airspace over Iran, Iraq, Kuwait, Israel and Bahrain virtually empty as Israel said it struck Iran and the US military initiated a series of strikes against targets in the country.
Iran retaliated with a salvo of missiles.
Airlines have cancelled almost 40 per cent of flights to Israel and 6.7 per cent of flights to the broader region on Saturday, according to preliminary Cirium data.
Witnesses told Reuters about explosions across the Gulf, including in Qatar's Doha, which hosts the biggest US military base in the Middle East, as well as Abu Dhabi and Dubai in the United Arab Emirates.
The escalation dimmed hopes for a diplomatic solution to Tehran's nuclear dispute with the west and reignited conflict after weeks of US military buildup in the region.
It marks the latest upheaval for air travel in the usually busy region amid escalating tensions.
Airports in the Middle East are among the busiest in the world, spanning from Iran and Iraq to the Mediterranean and serving as a hub for flights between Europe and Asia.
The region has also taken on a more important role since the start of the war between Russia and Ukraine, which has forced airlines to avoid airspace over both countries.
Conflict zones are an increasing operational burden on airlines as aerial attacks raise worries about accidental or deliberate shoot-downs of commercial air traffic.
Longer flight times also require more fuel, adding to their costs.
Israel, Iran, Iraq, Bahrain, Qatar, Kuwait and Jordan closed their airspace following the attacks and a map of the region on Flightradar24 showed planes avoiding these areas.
The European Union's aviation regulator EASA on Saturday recommended its airlines stay out of the airspace affected by the ongoing military intervention.
British Airways, owned by IAG, said it was monitoring the situation and had cancelled flights to Tel Aviv and Bahrain until March 3, as well as Saturday's flights to Amman.
The Russian Ministry of Transport said on Saturday that Russian air carriers had suspended flights to Iran and Israel.
Germany's Lufthansa said it was suspending flights to and from Dubai on Saturday and Sunday and halting temporarily the Tel Aviv, Beirut and Oman routes until March 7. Air France cancelled flights to and from Tel Aviv and Beirut.
Iberia also cancelled flights to Tel Aviv, while Wizz Air suspended flights to and from Israel, Dubai, Abu Dhabi and Amman with immediate effect until the same date, it said.
Passengers and airlines can expect airspace to be shut for some time in the region.
It's also important to look at the tensions between Afghanistan and Pakistan as that will limit airspace even further, said Eric Schouten, head of aviation security advisory Dyami.
"With hostilities now underway, the impact on regional aviation is immediate and highly fluid," he said.
"We also anticipate precautionary evacuations or temporary shutdowns at select Gulf airports if the threat envelope expands, which would immediately disrupt key transit hubs."
Various Indian airlines, including Air India, also suspended flights to the Middle East.
Regional airspace closures have disrupted several Emirates flights, the Dubai-based carrier said, while its sister airline flydubai said it had temporarily suspended operations on Saturday "due to the ongoing developments in the region".
Qatar Airways and Kuwait Airways temporarily suspended flights, while Turkish Airlines also cancelled flights to several Middle Eastern destinations.
