To investigate the antibiotic activity and synergistic effect, analogues were designed to increase not only net positive charge by Lys-substitution but also hydrophobic helix region by Leu-substitution from CA (1-8)-MA (1-12) hybrid peptide (CA-MA). In particular, CA-MA analogue P5 (P5), designed by flexible region (GIG-->P)-substitution, Lys- (positions 4, 8, 14, 15) and Leu- (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed potent antibacterial activity in minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC) without having hemolytic activity. In addition, P5 and chloramphenicol has potent synergistic effect against tested cell lines. As determined by propidium iodide (PI) staining, flow cytometry showed that P5 plus chloramphenicol-treated cells had higher fluorescence intensity than untreated, P5- and chloramphenicol-treated cells. The effect on plasma membrane was examined by investigating the transmembrane potential depolarizing experiments of S. aureus with P5 and chloramphenicol. The result showed that the peptide exerts its antibacterial activity by acting on the plasma membrane. Furthermore, P5 caused significant morphological alterations of S. aureus, as shown by scanning electron microscopy. Our results suggest that peptide P5 is an excellent candidate as a lead compound for the development of novel anti-infective agents and synergistic effects with conventional antibiotic agents but lack hemolytic activity.