In Greece, a National Monitoring Plan for Antimicrobial Resistance in sick bovine animals was launched (2021). We aimed to study the antimicrobial resistance profiles of 118 Staphylococcus aureus isolates obtained from dairy cows >18 months with mastitis in the Region of Central Macedonia from 1st March to 31st July 2021, where 45% (Census 2020: 69.186/128.511) of dairy cows in Greece are concentrated. By using the disk diffusion method (according to the CLSI guidelines), we tested susceptibility of isolates to nine antimicrobials (penicillin, cefoxitin, cefquinome, gentamicin, tetracycline, marbofloxacin, erythromycin, clindamycin, enrofloxacin) selected following the marketing amounts of animal use in Greece. In the study, 62 (52.54%) of S. aureus isolates were resistant to penicillin ; 57/118 were 48,31% resistant to erythromycin, 56/118 were resistant 47,46% to clindamycin; 29/118 were 24,58% resistant to tetracycline and cefquinome both, 27/118 were 22,88% resistant to cefoxitin, 20/118 were 16,95% resistant to gentamicin, 3/118 were 2,54% resistant to marbofloxacin and, 2/118 were 1,69% resistant to enrofloxacin. In relation to monitoring the antimicrobial resistance of Staphylococcus aureus isolates responsible for mastitis in cattle (where a combination of penicillin and an aminoglycoside is the predominant therapeutic treatment in Greece), additional research is required for beta-lactams (such as penicillin and cefoxitin). This investigation falls within the 'One Health' framework, addressing the judicious use of antibiotics in both human staphylococcal infections and veterinary medicine in Greece. However, it seems that several kinds of interventions at farm level led to a decrease in the quinolones use. The study offers a thorough understanding of the antimicrobial resistance patterns among Staphylococcus aureus isolates. The significant resistance rates to various antibiotics highlight the need for responsible antibiotic usage and continuous monitoring of resistance trends. These insights are crucial in developing effective strategies to monitor and combat the emergence and dissemination of resistant strains and ensuring better treatment and control of infections caused by this bacterium. The findings add valuable knowledge to our understanding of antibiotic resistance and its implications.