Antimicrobial peptides (AMPs) are an important component of innate immunity in both vertebrates and invertebrates, and some of the unique characteristics of AMPs are usually associated with their living environment. The marine fish, mudskipper Boleophthalmus pectinirostris, usually live amphibiously in intertidal environments that are quite different from other fish species, which would be an exceptional source of new AMPs. In the study, an AMP named Bolespleenin was identified, which was a truncated peptide derived from a new functional gene found in B. pectinirostris, that was up-regulated in response to bacterial challenge. Bolespleenin had only 14 amino acid residues, including five consecutive arginine residues. It was found that the peptide had broad-spectrum antibacterial activity, good thermal stability and sodium ion tolerance. Bolespleenin killed Acinetobacter baumannii and Staphylococcus aureus by disrupting the structural integrity of the bacterial membrane, leading to leakage of the cellular contents, and inducing accumulation of bacterial endogenous reactive oxygen species (ROS). In addition, Bolespleenin effectively inhibited biofilm formation of A. baumannii and S. aureus and long-term treatment did not lead to the development of resistance. Importantly, Bolespleenin maintained stable activity against clinically multi-drug resistant bacterial strains. In addition, it was noteworthy that Bolespleenin showed superior efficacy to LL-37 and vancomycin in a constructed mouse model of MRSA-induced superficial skin infections, as evidenced by a significant reduction in bacterial load and more favorable wound healing. This study provides an effective antimicrobial agent for topical skin infections with potential therapeutic efficacy for infections with drug-resistant bacteria, including MRSA.