Methicillin-resistant Staphylococcus aureus (MRSA) is a highly prioritized pathogen proposed by World Health Organization (WHO). It is listed to encourage researchers to search for effective antimicrobial agents. Previously, we isolated soil Brevibacillus sp. strain SPR19, which showed anti-MRSA activity. However, the active substances were still unknown. This study aimed to isolate and identify the anti-MRSA substances from this bacterium. The cell-free supernatant of SPR19 was subjected to salt precipitation, cation exchange, and re-versed-phase chromatography. Bioassay-guided fractionation was used to screen active sub-stances