Seduction Of The Innocent, easily. Not only did that book nearly destroy the industry, wipe EC Comics out of business, and force the remaining publishers to censor themselves in order to keep from suffering the same fate as EC, but it pretty much cemented the "comics are for kids" stigma that still exists to this day and was the forerunner of the constant censorship/legal problems comics still face today when stores get attacked for carrying books geared to adult readers.
I'd probably rank the creation of Superman and the instant popularity that followed his debut as #2 in terms of importance, but Wertham's negative impact on the industry had far greater consequences, whether we like it or not.
