Aim: The current study aimed to visualize the damage caused by enterolysin A to the cells of sensitive strains and to find out cleavage site within the peptidoglycan moiety of bacterial cell walls. Methods and Results: Enterolysin A produced by a local isolate, Enterococcus faecalis B9510 was found to rapidly kill cells of the sensitive strain Lactococcus lactis ssp.cremoris 2144 during 120min of treatment as compared to the untreated control where no such effect was observed. Transmission electron microscopy of the enterolysin A-treated cells revealed leaking of the cytoplasmic contents ultimately resulting in complete lysis of cell walls. To find the cleavage site, purified cell walls of L.lactis ssp.cremoris 2144, Pediococcus pentosaceus 43201 and Lactobacillus delbrueckii ssp.bulgaricus ATCC 11842 were treated with enterolysin A, and liberated amino acids were derivatized for N and C terminals and analysed using thin layer chromatography on silica gel with isopropanol as solvent. The results showed that enterolysin A cleaves the peptide bonds at two locations within peptidoglycan subunits. The first location is between L-alanine and D-glutamic acid of the stem peptide and the other location is between L-lysine of the stem peptide and D-aspartic acid of the interpeptide bridge. Conclusions: Enterolysin A cleaves the peptide bonds within the stem peptide as well as in the interpeptide bridge of Gram-positive bacterial cell walls. This gives a possible reason for the broad spectrum of enterolysin A activity. Significance and Impact of the Study: This is the first report identifying the cleavage site of enterolysin A within the cell walls of sensitive bacteria. This will help in identifying potential applications for enterolysin A.