Human drug metabolites (HDMs) are important chemicals widely used in drug-related studies. However, acquiring these enzyme-derived and regio-/stereo-selectively modified compounds through chemical approaches is complicated. PikC is a biosynthetic P450 enzyme involved in pikromycin biosynthesis from the bacterium Streptomyces venezuelae. Here, we identify the mutant PikCD50N as a potential biocatalyst, with a broad substrate scope, diversified product profile, and high catalytic efficiency, for preparation of HDMs. Remarkably, PikCD50N can mediate the drug-metabolizing reactions using the low-cost H2O2 as a direct electron and oxygen donor.