Antimicrobial peptides (AMPs) are promising alternative agents for treating multidrug-resistant bacterial infections. Aurein 1.2 is a natural 13-amino acid AMP with antibacterial activity against Gram-positive bacteria. In this study, we designed three novel AMPs: aurein M1 (A10W), aurein M2 (D4K, E11K), and aurein M3 (A10W, D4K, E11K) to analyze the effect of Trp substitution and enhancement of positive charge on the activity of aurein 1.2. The AMP probability, physicochemical properties, secondary and tertiary structures, and amphipathic structure were predicted by various bioinformatics tools. After the synthesis of the peptides, their antibacterial activity, hemolysis, cytotoxicity, and structural analysis were assayed. Compared to the selectivity of aurein 1.2, the selectivity of aurein M2 and M3 with a net positive charge of +5 was improved 11.30- and 8.00-fold against Gram-positive and -negative bacteria, respectively. The hemolytic activity of aurein M2 was lower than that of aurein 1.2 and M3, while the higher percentage of human fibroblast cells were alive in the presence of aurein M3. Also, the MICs of aurein M3 toward Staphylococcus aureus and Escherichia coli at the physiologic salt were ≤16, which is recommended as a promising candidate for clinical investigation. Circular dichroism analysis indicated an alpha-helical structure in the peptide analogs that is similar to aurein 1.2 in the presence of 10 mM SDS. Therefore, increasing positive charge can be used successfully as an approach for improving the potency and selectivity of AMPs. Moreover, the beneficial effect of Trp substitution depends on its position and the sequence of peptides