Severe infections due to highly virulent and resistant Staphylococcus aureus pose a serious health threat in Bulgaria and worldwide. The purpose of this study was to explore the clonal spread of recent clinically significant methicillin-susceptible S. aureus (MSSA) isolates from inpatients and outpatients treated in three university hospitals in Sofia, Bulgaria, during the period 2016-2020 and evaluate the relationship between their molecular epidemiology, virulence profiling, and antimicrobial resistance. A total of 85 isolates (invasive and noninvasive) were studied using RAPD analysis. Ten major clusters (A-K) were identified. The first major cluster A (31.8%) was found to be predominant during 2016 and 2017 and was widespread in two hospitals, unlike its case in the following years, when it was found to be replaced by newer cluster groups. All MSSA members of the second most common cluster F (11.8%) were recovered from the Military Medical Academy, mainly during 2018-2020, and were determined to be susceptible to all other groups of antimicrobials, except for penicillins without inhibitors because they harboured the blaZ gene. The newer cluster I, with 9.4% of the isolates absent in 2016-2017, showed significantly higher virulence and macrolide resistance (42.9%) due to ermB and ermC. All the isolated MSSA in groups F and I were nosocomial and mostly invasive. In conclusion, this 5-year study demonstrates the molecular epidemiology of MSSA infections in three Bulgarian hospitals. Findings can be helpful for the understanding of staphylococcal infection distribution in hospital settings and their prevention.