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Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is a severe threat to public health globally. The development of novel agents has encountered the repeated mechanism of drug resistance. This study aimed to investigate an anti-MRSA substance isolated from a promising soil bacterium. The result showed that an isolate (WUL10) was in the <i>Brevibacillus</i> genus. The minimum inhibitory concentration (MIC) of the purified substance was 1 µg/mL against <i>S. aureus</i> TISTR 517 and MRSA strains. This substance showed the bactericidal effect at the concentration of 1–2 µg/mL against these bacterial indicators. The activity of the substance retained more than 95% when encountering high temperatures and a wide range of pH, but it was sensitive to proteolytic enzymes and SDS. It was identified as a novel antimicrobial peptide (KVLVKYLGGLLKLAALMV-COOH) with the predicted structure of α-helix. The substance could rupture the cell wall of the tested pathogen. MIC and MBC of the synthesized peptide were 16 and 64 µg/mL, respectively. The difference in the activity between the isolated and synthetic peptides might be from the synergistic effects of other AMPs in the purified substance. This novel AMP would provide an advantage for further development of anti-MRSA substances to manage the situation of antibiotic resistance.