Many redox reactions are being catalyzed by oxidoreductase enzymes. Although they have high structural similarity, these enzymes undergo different reaction mechanisms and rates depending on the types of substrate, structure, and protonation state. Many pharmaceutical and food industry applications of these enzymes depend on the structural characterization and elucidation of the reaction mechanisms. Combined experimental studies with computational tools provided evidence on the isoalloxazine ring of the oxidoreductase’s flavin cofactor reacting through a step-wise process of consecutive single-electron transfers. Moreover, a reaction mechanism is enabled due to the protonation state of the proximity of the cofactor and substrate binding pocket. This paper aims to review the recent advances concerning the structural aspects and factors affecting the mechanism of reactions involving several representatives of the flavin-dependent oxidase family.