Staphylococcus spp. are one of the most predominant isolates in milk samples of dairy cows with mastitis worldwide. The aims of this study were to investigate the prevalence of bacterial pathogens in bovine mastitis milk samples in South Korea and the antimicrobial resistance profiles of staphylococcal isolates. In total, 1,245 strains were isolated from 1,260 mastitis quarter milk samples (with somatic cell counts ≥200,000 cells/mL) from 66 dairy farms between 2018 and 2022. The bacterial genus with the highest prevalence in bovine mastitis milk samples was Staphylococcus spp. (33.9%), followed by Streptococcus spp. (11.5%). S. aureus and non-aureus staphylococci (NAS) accounted for 11.0% and 89.0% of staphylococcal isolates, respectively. S. chromogenes was the most prevalent species among the 22 NAS species detected. S. aureus showed the highest resistance rates to penicillin (25.0%) and ampicillin (20.8%), whereas NAS showed the highest resistance rates to penicillin (18.3%), tetracycline (11.4%) and erythromycin (10.1%). Sixteen multidrug-resistant (MDR) isolates were only isolated from NAS, and the most commonly detected antimicrobial resistance gene in the 16 MDR isolates was mecA (75.0%), followed by tetK (62.5%), blaZ (50.0%), ermC (50.0%), and lnuA (43.8%). In conclusion, NAS were the most common isolates from mastitis milk in South Korea and MDR isolates carried a variety of antibiotic resistance genes. Our study suggests that continuous monitoring of the distribution and antimicrobial resistance in Staphylococcus spp., particularly NAS, is needed to improve the effectiveness of management and treatment strategies in dairy farms.