Monooxygenases are enzymes that catalyze the insertion of a single oxygen atom from O(2) into an organic substrate. In order to carry out this type of reaction, these enzymes need to activate molecular oxygen to overcome its spin-forbidden reaction with the organic substrate. In most cases, monooxygenases utilize (in)organic cofactors to transfer electrons to molecular oxygen for its activation. Monooxygenases typically are highly chemo-, regio-, and/or enantioselective, making them attractive biocatalysts. In this review, an exclusive overview of known monooxygenases is presented, based on the type of cofactor that these enzymes require. This includes not only the cytochrome P450 and flavin-dependent monooxygenases, but also enzymes that utilize pterin, metal ions (copper or iron) or no cofactor at all. As most of these monooxygenases require nicotinamide coenzymes as electron donors, also an overview of current methods for coenzyme regeneration is given. This latter overview is of relevance for the biotechnological applications of these oxidative enzymes.