This study aimed to investigate the spread of methicillin-resistant coagulase-positive staphylococci (MRCoPS) among veterinary staff, hand-touch sites and surgical tissue during cystotomy operations on cats and dogs that were patients, and to analyze the genetic relatedness and antimicrobial resistance profiles of the isolates. Human and environmental samples were obtained from the nasal passageways of 12 surgeons and veterinary assistants and from 29 hand-touch sites of instruments in operative units and subjected to bacterial isolation and enumeration. Swab samples were collected in triplicate from 29 dogs and three cats at the site of incision, from the incision area, from the peritoneum during surgery and from the peritoneum before suture. MRCoPS were identified by mecA gene detection and characterized by their antibiogram profile, SCCmec type and pulsed-field gel electrophoresis. Twenty-four staphylococci were isolated, derived from one veterinary assistant, 12 operating room floor areas and hand-touch sites, three dogs and one cat. Methicillin-resistant S. pseudintermedius (MRSP) was found on an electric clipper and rebreathing circuits in the operating room. Three dogs were positive for MRSP during surgery, and one methicillin-resistant S. aureus (MRSA) was detected in a cat. All MRCoPS were resistant to doxycycline, erythromycin, clindamycin and enrofloxacin, but no patients developed surgical site infections. According to their genotypic patterns, the clones obtained from the environment and human sources differed from the animal clones. Despite intensive hygienic management, a variety of MRCoPS clones were present within the surgical unit and during surgery.