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 30 min and 2 h 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 ± 0.35 μg/mL, whereas the mean value for MRSP was 0.47 ± 0.27 μg/mL. There was no difference between the mean MIC of MRSP and MSSP (P = 0.84). After a 30 min 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.