Belmonte touched in one minute, 59.61 seconds, more than 1.5 seconds ahead of Hosszu, who has dominated the short-course events in 2014.
It was the first world record of the championships in the Qatari capital, though the Spaniard had little time to savour the moment before she was back in the pool 40 minutes later for the 400 individual medley final.
Hosszu, who set the world record in the event last August and was five seconds faster than Belmonte in qualifying for the final, setting a championships record, again led from the front but tired in the freestyle leg and was chased down.
Belmonte clocked 4:19.86, beating Hosszu to the wall by more than three seconds and eclipsing the world record by almost a second. Britain's Hannah Miley, the 2012 world champion, was third in 4:24.74.
Hosszu, who has entered 10 events in Doha, said she felt having Belmonte in such good form would push her to better performances.
"It's good to have Mireia Belmonte here," she said. "Losing to her gives me extra motivation."
The Dutch women rounded off the record-breaking session when they clocked 7:32.85 for the relay, shattering the mark set by China in 2010 by more than three seconds.
France's men won the 4x100 freestyle title.
South Africa's Olympic 200 butterfly champion Chad le Clos, also surprised many to upset Ryan Lochte in the 200 freestyle final, winning in 1:41.45.
Russian's Danila Izotov was second, with Lochte, the most successful swimmer in the short course championships, in third after he qualified eighth for the final.
"It couldn't start any better, I was really happy to get the win," said le Clos.
"To get the first medal on the board, the 200m free is not my strongest event so I can only get better from here, touch wood!"
(Reporting by Greg Stutchbury in Wellington; Editing by Toby Davis)
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