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The US Open champion was unable to handle the lefthander, who attacked the Australian's backhand throughout the match to force errors, and lost in 70 minutes.
It was the second successive year the world number six has been the top seed at Brisbane and lost in the second round. Last year, she lost to compatriot Jarmila Gajdosova.
Benesova, who had never beaten Stosur in their previous four meetings, will now meet Australian Open champion Kim Clijsters in the quarter-finals.
Men's second seed Gilles Simon, however, had no such problems, dispatching Australia's James Duckworth 6-3 7-5 to move on to the quarter-finals.
The Frenchman joins Uzbekistan's Denis Istomin in the last eight, after he advanced courtesy of a walkover following Tommy Haas's withdrawal with a calf injury.
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