Flavoprotein monooxygenases create valuable compounds that are of high interest for the chemical, pharmaceutical, and agrochemical industries, among others. Monooxygenases that use flavin as cofactor are either single-or two-component systems. Here we summarize the current knowledge about two-component flavin adenine dinucleotide (FAD)-dependent monooxygenases and describe their biotechnological relevance. Two-component FAD-dependent monooxygenases catalyze hydroxylation, epoxidation, and halogenation reactions and are physiologically involved in amino acid metabolism, mineralization of aromatic compounds, and biosynthesis of secondary metabolites. The monooxygenase component of these enzymes is strictly dependent on reduced FAD, which is supplied by the reductase component. More and more representatives of two-component FAD-dependent monooxygenases have been discovered and characterized in recent years, which has resulted in the identification of novel physiological roles, functional properties, and a variety of biocatalytic opportunities.