To understand the influence of different hydrophobic amino acids at “a” and “d” positions of a heptad repeat sequence on antimicrobial, cytotoxic, and antiendotoxin properties, four 15-residue peptides with leucine (LRP), phenylalanine (FRP), valine (VRP), and alanine (ARP) residues at these positions were designed, synthesized, and characterized. Although valine is similarly hydrophobic to leucine and phenylalanine, VRP showed significantly lesser cytotoxicity than LRP and FRP; further, the replacement of leucines with valines at “a” and “d” positions of melittin-heptads drastically reduced its cytotoxicity. However, all four peptides exhibited significant antimicrobial activities that correlate well with their interactions with mammalian and bacterial cell membranes and the corresponding lipid vesicles. LRP most efficiently neutralized the LPS-induced pro-inflammatory mediators like NO, TNF-α, and IL-6 in macrophages followed by FRP, VRP, and ARP. The results could be useful for designing short antimicrobial and antiendotoxin peptides with understanding the basis of their activity.