Antibiotic-resistant bacteria are becominging increasingly common, posing a dire threat to global health, complicating the treatment of common infections and the performance of routine medical procedures. As a result, it is more important than ever to invest in research aimed at discovering novel, effective antimicrobial agents. One promising class of agents is antimicrobial peptides (AMPs), which are naturally occuring or synthetic peptides that exhibit potent antimicrobial bioactivity through a wide array of mechanisms. While these antimicrobial characteristics make AMPs a promising clinical antibiotic, unfortunately, AMPs often display significant cytotoxicity and hemolytic activity which limits their medical applications. Aurein-1.2 is a natural occuring AMP synthesized by Litoria raniformis (the Southern Bell Frog), which has demonstrated strong antimicrobial and hemolytic activity (Ramezanzadeh 2020). In this study four derivatives of Aurein-1.2 (T1, T2, T3, and T4) were synthesized by substituting two lysine residues with arginine and two isoleucine residues with tryptophan at varying positions. To analyze the antimicrobial activity of the derivatives, a Minimum Inhibitory Concentration (MIC) assay was performed using the peptides at varying concentrations against Escherichia coli and multidrug resistant Escherichia coli, Salmonella typhimurium, Candida albicans, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA). The results showed that T1 and T2 exhibit >90% cell death at 50 μg/ml , while T3 displayed similar activity at the same concentrations against all bacteria except for Escherichia coli, which required 100 μg/ml . T4 required 100 μg/ml for for >90% cell death for all tested organisms. Hemolytic activity against mammalian erythrocytes was also analyzed in this study using hemolytic assays with whole horse blood. Cytoxicity was observed at all tested concentrations in all derivaties except for T1 and T3 at 10 μg mL-1 .