A 5-year-old, 8.95 kg, female Schnauzer presented anorexia with a 3-day history and increased heart sound intensity. Based on the clinical and echocardiographic findings along with the positive blood culture result, the dog was diagnosed with infective endocarditis (IE). Using proper antibiotics treatment, clinical signs were improved within 3 days and resolved within 1 week. For exact identification of the causative agent, multiplex polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) methods were performed. The etiological agent was confirmed as Staphylococcus pseudintermedius with antibiotics resistance genes such as β-lactamase (blaZ) and methicilline resistance (mecA). The bacterial virulence factors included pyogenic toxin genes such as staphylococcal enterotoxins A, B, C, D, and E and toxic shock syndrome toxin 1. Diagnosis of IE is challenging due to a variety of non-specific clinical presentations, rapid disease progression, and lack of a confirmative diagnostic technique. This report demonstrated that such molecular diagnostics could be very useful for diagnosing and identifying characteristics of the causative organism for prediction of prognosis and proper treatment. To our knowledge, this is the first report on the isolation of S. pseudintermedius using molecular diagnostics from a clinical case of canine IE.