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Biedermann clocked one minute 42 seconds to beat Phelps' Beijing Olympic mark of 1:42.96.
The American, 1.22 seconds slower, had to settle for a silver, with Russian Danila Izotov third in 1:43.90.
"The suits make a difference," said Biedermann, who was virtually unknown before the worlds.
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Phelps (front) and Biedermann are at the awarding ceremony of the world swimming championships in Rome, July 28, 2009 |
"I hope there will be a time when I can beat Michael Phelps without these suits," the German added.
Biedermann opened another night of record breaking. Four more world marks were rewritten, raising the total to 15 through the first three days of an eight-day meet.
Britain's Gemma Spofforth set the world record to win the 100m backstroke, while Italy's Federica Pellegrini electrified the home crowd with her second world record of the championships in a semifinal heat of the 200 freestyle.
South African Cameron van der Burgh bettered the 50m breaststroke world standard in the semifinals.
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Phelps (front) and Biedermann are at the awarding ceremony of the world swimming championships in Rome, July 28, 2009. |
Russian Yuliya Efimove got the silver and Kasey Carlson of the United States was third.
Japanese Junya Koga won the 100m backstroke final in 52.26, beating Helge Meeuw of Germany and Spain's Aschwin Grigoriadis.
Italy's Alessia Filippi won the 1,500m freestyle with Denmark's Lotte Friis taking the silver and Romania's Camelia Potec the bronze.
Spofforth won the 100m backstroke in 58.12 seconds, beating the mark of 58.48 set a day earlier by Anastasia Zueva.
Zueva picked the silver and Australian Emily Seebohm got the bronze.