This study assessed the production of a bacteriocin-like substance by Staphylococcus pseudintermedius S28, and evaluates its inhibitory effect against isolates of S. aureus from foods. All indicator isolates were sensitive to the substance produced from S. pseudintermedius S28, showing growth inhibition zones ranging from 14.2 to 28.3 mm. The inhibitory substance has no effect against the producer strain. The inhibitory substance was affected by proteolytic enzymes, while glycolytic and lipolytic enzymes had no effect, suggesting that the active substance could be considered as a bacteriocin-like substance. From these results, S. pseudintermedius S28 could be an interesting producer of a bacteriocin-like substance capable of strongly inhibiting S. aureus.