Mastitis is an inflammatory condition of the mammary tissue in bitches, most common during the postpartum period and pseudopregnancy, and it significantly contributes to neonatal mortality. In the present study, thirty-nine bitches aged 1 to 12 years were examined, and 156 milk samples were collected. Cases were classified as either clinical mastitis (CM), characterized by overt local signs with or without systemic manifestations, or intramammary infection (IMI), defined as bacteriological culture positivity in the absence of clinical signs. Conventional culture and biochemical tests were used to identify bacterial species and assess antimicrobial resistance patterns. The study specifically aimed to investigate the resistance characteristics and biofilm-forming ability of Staphylococcus aureus (S. aureus) isolates obtained from canine milk. Molecular confirmation of S. aureus was performed by amplification of the nucA gene. Antimicrobial susceptibility was assessed using the disk diffusion method, and eight resistance-associated genes were detected via polymerase chain reaction (PCR). Biofilm production was assessed by a microtiter plate assay. Of all milk samples, 56.41 % yielded positive bacterial growth. S. aureus was the most frequently isolated species, accounting for 55.68 % of positive cultures. Among the S. aureus isolates, 51.02 % met the criteria for multidrug resistance, defined as resistance to at least one agent in three or more antimicrobial classes. The most commonly detected resistance gene was blaZ, followed by ermA, ermB, and ermC. Over half of the S. aureus isolates demonstrated measurable biofilm-forming ability. These findings underscored the clinical importance of S. aureus in canine mastitis and highlighted the necessity of routine bacteriological culture, antimicrobial susceptibility testing, and molecular diagnostics to guide effective treatment strategies and improve health outcomes in affected bitches and their offspring

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