Dimethyl 4,4′ -(O-phenylene)bis(3-thioallophanate), commonly known as methyl thiophanate (MT), is a systemic fungicide and suspected carcinogen to humans. In this study, the oxidative potential of this category-III acute toxicant has been ascertained based on its capacity of inducing reactive oxygen species (ROS) and promutagenic 8-oxo-7,8-dihydro-2′ -deoxyguanosine (8-oxodG) adducts in DNA. The discernible MT dose-dependent reduction in fluorescence intensity of a cationic dye rhodamine (Rh-123) in human lymphocytes and increased fluorescence intensity of 2′,7′-Dichlorodihydro fluorescein diacetate (DCFH-DA) treated cells signifies decreased mitochondrial membrane potential (Δ Ψ m) due to intracellular ROS generation. The ³²P-post-labeling assay demonstrated the MT-induced 8-oxodG adduct formation in calf thymus DNA. Thus, it is concluded that MT, as a potent oxidative stressor, produces ROS leading to mitochondrial dysfunction, oxidative DNA damage and mutagenesis.