Structural characterization of BMAP-28(1-18), a potent bovine myeloid antimicrobial peptide can aid in understanding its mechanism of action at molecular level. We report NMR structure of the BMAP-28(1-18) and its mutated analogue mutBMAP18 in SDS micelles. Structural comparison of the peptides bound to SDS micelles and POPE-POPG vesicles using circular dichroism, suggest that structures in the two lipid preparations are similar. Antimicrobial assays show that even though both these peptides adopt helical conformation, BMAP-28(1-18) is more potent than mutBMAP18 in killing bacterial cells. Our EM images clearly indicate that the peptides target the bacterial cell membrane resulting in leakage of its contents. The structural basis for difference in activity between these peptides was investigated by molecular dynamics simulations. Inability of the mutBMAP18 to retain its helical structure in presence of POPE:POPG membrane as opposed to the BMAP-28(1-18) at identical peptide/lipid ratios could be responsible for its decreased activity. Residues SerS, Arg8 and Arg12 of the BMAP28(1-18) are crucial for its initial anchoring to the bilayer. We conclude that along with amphipathicity, a stable secondary structure that can promote/initiate membrane anchoring is key in determining membrane destabilization potential of these AMPs. Our findings are a step towards understanding the role of specific residues in antimicrobial activity of BMAP-28(1-18), which will facilitate design of smaller, cost-effective therapeutics and would also help prediction algorithms to expedite screening out variants of the parent peptide with greater accuracy.