Staphylococcus pseudintermedius, commonly involved in canine pyoderma, can be classified as meticillin-susceptible S.pseudintermedius (MSSP) or meticillin-resistant S.pseudintermedius (MRSP). MRSP infections may be difficult to treat due to broad β-lactam resistance of MRSP and typically additional multidrug-resistance. Topical antibacterial treatment is the preferred treatment modality for surface and superficial skin infections. HYPOTHESIS⁄OBJECTIVES: Mangosteen crude extract containing the antibacterial compound α-mangostin will have in vitro activity against MSSP and MRSP isolated from canine pyoderma. Twenty-three samples, MSSP (n=12) and MRSP (n=11), isolated from canine pyoderma. Minimum inhibitory concentrations (MICs) were determined for mangosteen crude extract by broth microdilution. High-performance liquid chromatography (HPLC) analysis was used to determine the amount of α-mangostin in mangosteen crude extract. A time-kill assay was performed at 30min and 2h after exposure to a high concentration of crude extract (100× MIC). Antibacterial activity for α-mangostin was calculated according to HPLC results. The concentration of α-mangostin was 17.72±1.42% w/w. The mean MIC of α-mangostin towards MSSP was 0.53±0.35μg/mL, whereas the mean value for MRSP was 0.47±0.27μg/mL. There was no difference between the mean MIC of MRSP and MSSP (P=0.84). After a 30min exposure to 100× MIC of the crude extract, a 95% reduction in colony forming units was found. The results showed that α-mangostin in mangosteen crude extract was effective in inhibiting S.pseudintermedius (both MRSP and MSSP). Clinical studies are needed to investigate this effectiveness further in vivo.