Flight maps showed airspace over Iran, Iraq, Kuwait, Israel and Bahrain virtually empty after the strikes began, and Iran retaliated with missiles.
Blasts were reported in Qatar, home to the biggest US military base in the region, as well as in Abu Dhabi and Dubai, key east-west transit hubs.
"You have crews, planes and passengers stranded all over the world. It's a massive logistical nightmare," a Gulf airline source said.
Dubai Airports suspended all flights at Dubai International and at Al Maktoum International until further notice, urging passengers not to travel.
Emirates and flydubai temporarily halted operations, while Etihad suspended all departures from Abu Dhabi until 1000 GMT (9pm AEDT) on Sunday.
Students travelling from Paris to Dubai said their university trip was abandoned.
"We still have some students that went there earlier and they're stuck in Dubai and we don't know when they'll be able to come back," said Benjamin Gnatek.
At Charles de Gaulle airport, Thai-bound traveller Roman Simon said his onward flight via Doha was cancelled.
"Now, we're trying to find a solution to still make our trip to Thailand," he told Reuters.
At Doha's Hamad International Airport, gates were nearly empty as stranded passengers queued to make hotel arrangements, a Reuters witness said.
As countries in the region closed their airspace, aircraft were forced to divert around Larnaca, Jeddah, Cairo and Riyadh.
Flightradar24 briefly went down due to surging demand.
The escalation dimmed hopes for progress on Tehran's nuclear dispute with the west and reignited conflict after weeks of US military buildup.
"Passengers and airlines can expect airspace to be shut for quite some time," said Eric Schouten, head of aviation security advisory Dyami.
Airlines cancelled on Saturday about half of their flights to Qatar and Israel and about 28 per cent of their flights to Kuwait, after the United States and Israel launched strikes on Iran, according to preliminary Cirium data.
That number seems likely to rise.
In total, about 24 per cent of flights to the Middle East were cancelled, the data showed.
The region has become more important for global aviation since the Russia-Ukraine war forced airlines to avoid both countries' airspace.
Conflict zones add to operational risks, raising fears of accidental shoot-downs and lengthening routes, which increases fuel costs.
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 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 the Tel Aviv, Beirut and Oman routes until March 7.
Air France and Iberia also cancelled flights to Tel Aviv and Beirut, while Wizz Air suspended flights to Israel, Dubai, Abu Dhabi and Amman.
India put carriers on alert as Air India and IndiGo suspended services.
Hong Kong's Cathay Pacific Airways suspended operations in the region, affecting passenger flights to and from Dubai and Riyadh, as well as freighter services operating through Al Maktoum airport in Dubai.
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