The primary protagonist of Something Rotten is Thursday Next, who is the main protagonist of the series of the same name. Thursday Next is a "literary detective," solving crimes involving literature and literary heroes. In her world, the border between reality and fiction is much thinner than it is in ours, and Thursday is able to enter stories and interact with the characters in them. For example, Hamlet is also another important character in Something Rotten, although he is absent for much of the book. Thursday's family, including her parents and her young son (Friday Next), also feature heavily in the novel. Additionally, alternate versions of Thursday herself make appearances.

The primary antagonist of Something Rotten is Yorrick Kaine, a previously fictional character who has entered the real world and has high ambitions of leadership. In Something Rotten, he joins forces with an evil megacorporation and plots to become the Prime Minster. Thursday must defeat Yorrick and the Goliath corporation to prevent the ultimate end of the world.