Antimicrobial peptides (AMPs) are relatively short peptides that have the ability to penetrate the cell membrane, form pores leading to cell death. This study compares both antimicrobial activity and cytotoxicity of native melittin and its two mutants, namely, melittin I17K (GIGAVLKVLTTGLPALKSWIKRKRQQ) with a higher charge and lower hydrophobicity and mutant G1I (IIGAVLKVLTTGLPALISWIKRKRQQ) of higher hydrophobicity. The antimicrobial activity against different strains of Listeria was investigated by bioassay, viability studies, fluorescence and transmission electron microscopy. Cytotoxicity was examined by lactate dehydrogenase (LDH) assay on mammalian Caco-2 cells. The minimum inhibitory concentration of native, mutant I17K, mutant G1I against Listeria monocytogenes F4244 was 0.315±0.008, 0.814±0.006 and 0.494±0.037μg/ml respectively, whereas the minimum bactericidal concentration values were 3.263±0.0034, 7.412±0.017 and 5.366±0.019μg/ml respectively. Lag time for inactivation of L. monocytogenes F4244 was observed at concentrations below 0.20 and 0.78μg/ml for native and mutant melittin I17K respectively. The antimicrobial activity against L. monocytogenes F4244 was in the order native>G1I>I17K. Native melittin was cytotoxic to mammalian Caco-2 cells above concentration of 2μg/ml, whereas the two mutants exhibited negligible cytotoxicity up to a concentration of 8μg/ml. Pore formation in cell wall/membrane was observed by transmission electron microscopy. Molecular dynamics (MD) simulation of native and its mutants indicated that (i) surface native melittin and G1I exhibited higher tendency to penetrate a mimic of bacterial cell membrane and (ii) transmembrane native and I17K formed water channel in mimics of bacterial and mammalian cell membranes.