Broad spectrum activities, a unique mode of actions and rare resistant variants make antimicrobial peptide (AMP) a potential alternative to antibiotics. However, AMPs still have limitations in clinical development due to their physiological stability, toxicity and manufacturing costs. Cbf-14, derived from cathelicidin-BF has been proven to be effective against drug-resistant bacteria. Herein, a series of Cbf-14 mutants were designed to overcome these limitations. Design strategies included substitutions of lysine (Lys) or leucine (Leu) with similar residues such as ornithine (Orn) and norleucine (Ile), which are unnatural amino acids, to generate AMPs with enhanced therapeutic potential. Antimicrobial activity, hemolytic activity and cytotoxicity against mouse spleen cells of the peptide mutants were investigated. Among all of the mutants, Cbf-14-2 was regarded as the most potent candidate with the amino acid sequence of RLLR-Orn-FFR-Orn-LKKSV-NH2, which exhibited a superior antimicrobial activity with a minimum inhibitory concentration (MIC) of 4–32 μg/ml. Meanwhile, Cbf-14-2 displayed low levels of hemolysis in sheep red blood cells (sRBCs) and negligible cytotoxicity against mouse spleen cells, suggesting low toxicity against mammalian cells. A circular dichroism (CD) study indicated that Cbf-14-2 has a higher alpha-helix content than Cbf-14 (68.3% vs 35.1%) in SDS, which may contribute to its superior activity. Time-killing curves showed Cbf-14-2 can eliminate all tested bacteria within 240 min, suggesting its rapid bactericidal effect. Transmission electron microscopy (TEM), flow cytometry and calcein release assays revealed its excellent antimicrobial potency by inducing membrane permeation and disruption. In addition, Cbf-14-2 (10 mg/kg) could significantly elevate the survival rate of clinical strain infected mice, with a survival rate of 70.0%. Taken together, the data suggest that Cbf-14-2 possesses effective antimicrobial activity against penicillin-resistant bacteria in vitro and in vitro, thus rendering it as a potential anti-infective agent in clinical settings.