Bethoxazin is a broad spectrum industrial biocide with commercial applications as a material preservative; however its mechanism of action has not been investigated. In this study, the chemical reactivity of bethoxazin towards biologically important nucleophiles was assessed with UV-Vis spectroscopy. Bethoxazin reacted with molecules containing free sulfhydryl groups such as glutathione and human serum albumin but not with amino, acetate or phenol containing compounds. Bethoxazin was shown to potently inhibit the growth of the K562 human cancer cell line with an IC50 value in the micromolar range. The sulfydryl fluorescent label ThioGlo-1 was used to investigate the biological effects of bethoxazin in K562 cells and explore its mechanism of action. Bethoxazin inhibited the formation of covalent adducts in K562 cells between the free sulfhydryl group of biomolecules and ThioGlo-1, implying that bethoxazin reacts with molecules containing free sulfhydryl groups. Likewise, when glutathione was depleted in K562 cells, by buthionine sulfoximine, high concentrations of bethoxazin were able to inhibit the formation of covalent adducts between sulfhydryl biomolecules and ThioGlo-1. The growth inhibition assay (MTS) was used to investigate the effect of continuous bethoxazin treatment versus wash out in K562 cells. The MTS assay revealed a reduction in the potency of bethoxazin due to the wash out effect, suggesting that the growth inhibition effects of bethoxazin are likely not due to glutathione depletion. A two-colour flow cytometry analysis of bethoxazin treated K562 cells for eight hours demonstrated that bethoxazin provokes necrosis induced cell death in K562 cells. Taken together, these experimental results demonstrate that the reaction of bethoxazin with proteins containing an accessible sulfhydryl group is more likely to be the mechanism of action of the cell growth inhibition effects rather than glutathione depletion.