Sophoraflavanone B (SPF-B), a prenylated flavonoid, can be isolated from the roots of Desmodium caudatum. The aim of this study was to determine the mechanism of SPF-B's antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). MRSA is a multidrug-resistant pathogen and the main cause of hospital- and community-acquired infections. The minimum inhibitory concentration (MIC) of SPF-B was assessed using the broth microdilution method. The mechanism of action of SPF-B on S. aureus was analyzed in combination assays incorporating detergents, ATPase inhibitors, and peptidoglycan (PGN) derived from S. aureus. Furthermore, morphological changes in the SPF-B-treated MRSA strains were investigated using transmission electron microscopy. The MIC of SPF-B for MRSA was in the range of 15.6-31.25 μg/mL. The mechanism of action of SPF-B on MRSA was investigated using combination assays with detergent and ATPase inhibitors. The optical density at 600 nm of MRSA suspensions treated with a combination of detergent and SPF-B reduced the MRSA by 63%-73%. In the SPF-B and PGN combination assay, direct binding of SPF-B with PGN from S. aureus was evident. These data may be validated for the development of new antibacterial drugs for low MRSA resistance.