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Background: A collection of 400 enterococcal isolates from clinical samples of hospitalized patients were studied for their virulence traits according to the isolation site. Formation of biofilm and production of DNase, hemolysin, lipase, and gelatinase were characterized. Biofilms of selected strains were examined for their susceptibility to antimicrobial photodynamic therapy (aPDT). Methods: All strains were tested for biofilm production by microtiter method and the activity of hemolysin, gelatinase, lipase, and DNase by plate method with an adequate substrate. Photodynamic therapy with Photolon and red laser light was performed towards 48 h biofilms of eight representative strains. The viability of biofilms was tested by the BactLight assay and visualized under a fluorescent microscope. Results: Among the 400 isolates, 69.8% strains of Enterococcus faecalis, 30% of Enterococcus faecium, and 0.2% of Enterococcus casseliflavus were identified. In vitro, production of biofilm was found in 65.7% of enterococci. Biofilm-positive strains were isolated from urinary tract infections (81%), wound infections (71%), respiratory tract infections (62%), and gastrointestinal tract (47%) (colonization). Hemolysin activity was observed in 28.5%, gelatinase in 24.5%, lipase in 23%, and DNase in 3.5% of all, mostly biofilm-positive, isolates. Photodynamic therapy with Photolon efficiently reduced the enterococcal biofilms. Conclusions: The study demonstrated the high prevalence of biofilm-producing clinical enterococci, their virulence potential being higher than for biofilm-negative strains, and the susceptibility to aPDT of mature biofilms produced by strains, regardless of their species and site of isolation. [ABSTRACT FROM AUTHOR]