Flavin-containing monooxygenase (FMO) oxygenates drugs/xenobiotics containing a soft nucleophile through a C4a hydroperoxy-FAD intermediate. Human FMOs 1, 2 and 3, expressed in Sf9 insect microsomes, released 30–50% of O2 consumed as H2O2 upon addition of NADPH. Addition of substrate had little effect on H2O2 production. Two common FMO2 (the major isoform in the lung) genetic polymorphisms, S195L and N413K, were examined for generation of H2O2. FMO2 S195L exhibited higher “leakage”, producing much greater amounts of H2O2, than ancestral FMO2 (FMO2.1) or the N413K variant. S195L was distinct in that H2O2 generation was much higher in the absence of substrate. Addition of superoxide dismutase did not impact H2O2 release. Catalase did not reduce levels of H2O2 with either FMO2.1 or FMO3 but inhibited H2O2 generated by FMO2 allelic variants N413K and S195L. These data are consistent with FMO molecular models. S195L resides in the GxGxSG/A NADP+ binding motif, in which serine is highly conserved (76/89 known FMOs). We hypothesize that FMO, especially allelic variants such as FMO2 S195L, may enhance the toxicity of xenobiotics such as thioureas/thiocarbamides both by generation of sulfenic and sulfinic acid metabolites and enhanced release of reactive oxygen species (ROS) in the form of H2O2.