Escherichia coli O157:H7 contamination is a significant meat safety issue in many countries. Allyl isothiocyanate (AIT) is a natural compound found to limit the survival of E. coli O157:H7 and other pathogens in meat and meat products. In the present study, it was found that glutathione and cysteine naturally present in meat can interfere with AIT antimicrobial activity. Spectroscopy, HPLC, and LC-MS were used to confirm that glutathione was able to react with AIT and formed a conjugate with no or low bactericidal activity against the tested organisms. The same reaction also occurred at pH values of 4.9 and 5.8 at 25 and 4 degrees C, respectively, which broadly represent storage conditions in raw beef (pH 5.8) and during fermented sausage (pH 4.9) manufacture. Reactions observed help to explain reduction in antimicrobial potency of AIT in food (meat) systems.