Designing short antimicrobial peptides (AMPs), which are active against drug-resistant bacteria, is a promising way to find new therapeutic agents. In this research, a novel short AMP, PL-101-WK, was designed based on PL-101 (a derivative of plicatamide). Here, the substitution of Phe and His with Trp and Lys was considered. The antimicrobial activity and physicochemical properties of PL-101-WK were compared with PL-101 by in silico analysis. The antimicrobial activity in the presence or absence of NaCl concentration, thermal stability, hemolytic activity, and selectivity of the peptides were determined. By substitution of Lys and Trp residues, positive charge, in vitro stability, and hydrophilicity of PL-101-WK were raised compared to the template. PL-101-WK had the best minimum inhibitory concentration (MIC) value of 64 μg/ml against Staphylococcus aureus strains, which showed at least 16-fold reduction when compared to the values of PL-101. The MICs of PL-101-WK were retained toward S. aureus strains at physiological salt concentration. While PL-101-WK did not display acceptable thermal stability, it had desirable selectivity against bacteria. The maximum hemolytic activity of PL-101-WK was 1.65% at 512 μg/ml. Taken together, increasing positive charge and the presence of Trp residues were enhanced the potential of antibacterial activity of PL-101.