The emergence of methicillin-resistant Staphylococcus aureus (MRSA) as a highly pathogenic strain in veterinary and human medicine is a growing global problem. This study aimed to evaluate MRSA isolates of human and animal origin against various antibiotics in Yogyakarta, Indonesia. The susceptibility test was carried out by the disk diffusion method using Mueller-Hinton agar against nine antibiotic disks. Methicillin-resistant S. aureus strains were genetically confirmed through mecA gene detection encoding for methicillin resistance by polymerase chain reaction. All 240 S. aureus strains isolated from animals and humans were resistant to penicillin G (P) (100% and 99%, respectively), followed by ampicillin (AMP), amoxicillin (AML), oxacillin (OX), erythromycin (E), clindamycin (DA), tetracycline (TE), gentamicin (GEN), and ciprofloxacin (CIP). Eighty-three MRSA strains were resistant to OX (100%), P (100%), AMP (99.27%), AML (95.52%), E (87.77%), TE (71.33%), DA (63.24%), GEN (38.81%), and CIP (26.87%). The antimicrobial resistance pattern of S. aureus human isolates was similar to their animal counterpart, with 77.20% of MRSA strains classified as multidrug-resistant (MDR) bacteria. These findings indicate an increase in MDR S. aureus strains of animal origin in Yogyakarta, thus raising public health concerns about MRSA zoonotic spread.