Lol Bowser? I guess the list goes by longevity in the gaming industry instead of uniqueness and actual villainy. Andrew Ryan would be my top pick. The atmosphere of Rapture itself adds to Ryan's evilness, but he was a sick guy. The joker and The Reapers would round out my top 3.
I'm going with intelligence, ability to carry out a plot/plan, and overall effectiveness. Bowser never even killed/got away with the princess so how is he the best villian? The reapers killed millions of people, Andrew Ryan brought down a civilization, and the Joker fucked with everyone, how are they below freakin' Bowser?!?!?!
Opinionated list is opinion