This study aimed to evaluate the frequency of isolation of Staphylococcus aureus from different pathological samples processed in the Microbiology Laboratory of the National Institute of Infectious Diseases “Prof. Dr. Matei Balș”, Romania, between 1 January 2017 and 31 December 2022, aiming to establish the ratio of methicillin-resistant to methicillin-susceptible Staphylococcus aureus strains and the antibiotic resistance pattern of isolated microorganisms. The data of isolates originating from routine diagnostic tasks were analyzed retrospectively using laboratory data from the microbiology department. Up to 39.11% of Staphylococcus aureus strains were resistant to oxacillin (MRSA), with 49.97% resistance to erythromycin and 36.06% inducible resistance to clindamycin. Resistance rates to ciprofloxacin, rifampicin, gentamicin, and trimethoprim-sulfamethoxazole were 9.98%, 5.38%, 5.95%, and 0.96%, respectively. There was no resistance to vancomycin. Between 2017 and 2022, the percentage of MRSA strains decreased from 41.71% to 33.63%, sharply increasing to 42.42% in 2021 (the year of the COVID-19 pandemic, when the percentage of strains isolated from lower respiratory tract infections was higher than that of strains isolated from wounds or blood, as in previous years). This study showed a high percentage of MRSA strains (39.11% overall) with a higher proportion of these strains isolated from the blood (42.49%) compared to other clinical specimens.