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Nisin is a bacteriocin produced by many strains of Lactococcus lactis. This study examined the effect of nisin on Mycobacterium smegmatis, a non-pathogenic species of Mycobacterium. Nisin had a minimum inhibitory concentration of 8.0 μg ml−1 and a minimum inhibitory dose of 7.5 μg ml−1 against Myco. smegmatis. Treatment with 25.0 μg ml−1 nisin caused partial inhibition of Myco. smegmatis; the survivors were nisin-sensitive when tested in a separate experiment. Mycobacterium smegmatis cells exposed to 50.0 μg ml−1 of nisin, lost their viability. The effect of nisin on the growth of Myco. smegmatis was both time- and concentration-dependent. Nisin (10.0 μg ml−1) caused 97.7 ± 2.0% reduction in internal ATP and leakage of intracellular ATP out of Myco. smegmatis cells after several hours of treatment. These data suggest that nisin inhibits Myco. smegmatis by the same mechanism by which it inhibits other bacteria and warrants further investigation as a possible antitubercular agent.