ABSTRACTWe characterized the population ofStaphylococcus aureusfrom patients with atopic dermatitis (AD) in terms of (i) genetic diversity, (ii) presence and functionality of genes encoding important virulence factors: staphylococcal enterotoxins (sea,seb,sec,sed), toxic shock syndrome 1 toxin (tsst-1), and Panton-Valentine leukocidin (lukS/lukF-PV) by spa typing, PCR, drug resistance profile determination, and Western blot. We then subjected the studied population ofS. aureusto photoinactivation based on a light-activated compound called rose bengal (RB) to verify photoinactivation as an approach to effectively kill toxin-producingS. aureus. We have obtained 43 differentspatypes that can be grouped into 12 clusters, indicating for the first-time clonal complex (CC) 7 as the most widespread. A total of 65% of the tested isolates had at least one gene encoding the tested virulence factor, but their distribution differed between the group of children and adults, and between patients with AD and the control group without atopy. We detected a 3.5% frequency of methicillin-resistant strains (MRSA) and no other multidrug resistance. Despite genetic diversity and production of various toxins, all isolates tested were effectively photoinactivated (bacterial cell viability reduction ≥ 3 log10units) under safe conditions for the human keratinocyte cell line, which indicates that photoinactivation can be a good option in skin decolonization.IMPORTANCEStaphylococcus aureusmassively colonizes the skin of patients with atopic dermatitis (AD). It is worth noting that the frequency of detection of multidrug-resistantS. aureus(MRSA) in AD patients is higher than the healthy population, which makes treatment much more difficult. Information about the specific genetic background ofS. aureusaccompanying and/or causing exacerbations of AD is of great importance from the point of view of epidemiological investigations and the development of possible treatment options.