Antimicrobial resistance inStaphylococcusspecies from companion animals is becoming increasingly prevalent worldwide.S. pseudintermediusis a leading cause of skin infections in companion animals. α-mangostin (α-MG) exhibits various pharmacological activities, including antimicrobial activity against G (+) bacteria. This study investigated the antimicrobial activity of α-MG against clinical isolates ofStaphylococcusspecies from companion animals and assessed the therapeutic potential of α-MG in skin diseases induced byS. pseudintermediusin a murine model. Furthermore, the action mechanisms of α-MG againstS. pseudintermediuswere investigated. α-MG exhibited antimicrobial activity against clinical isolates of five differentStaphylococcusspecies from skin diseases of companion animalsin vitro, but not G (-) bacteria. α-MG specifically interacted with the major histocompatibility complex II analogous protein (MAP) domain-containing protein located in the cytoplasmic membrane ofS. pseudintermediusvia hydroxyl groups at C-3 and C-6. Pretreatment ofS. pseudintermediuswith anti-MAP domain-containing protein polyclonal serum significantly reduced the antimicrobial activity of α-MG. The sub-minimum inhibitory concentration of α-MG differentially regulated 194 genes, especially metabolic pathway and virulence determinants, inS. pseudintermedius. α-MG in pluronic lecithin organogel significantly reduced the bacterial number, partially restored the epidermal barrier, and suppressed the expression of cytokine genes associated with pro-inflammatory, Th1, Th2, and Th17 in skin lesions induced byS. pseudintermediusin a murine model. Thus, α-MG is a potential therapeutic candidate for treating skin diseases caused byStaphylococcusspecies in companion animals.