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<i>Enterococcus faecalis</i> and <i>E. faecium</i> are opportunistic pathogens commonly found in the microbiota of humans and other animals as well as in the environment. This article presents the results of antimicrobial susceptibility testing using phenotypic methods (broth microdilution and standardized disk diffusion) on selected clinical, food, and wastewater isolates of <i>E. faecalis</i> and <i>E. faecium.</i> The isolates were divided into subgroups based on their sensitivity to the following antibiotics: vancomycin (VAN) and ciprofloxacin (CIP), and biocides triclosan (TCL) and chlorhexidine (CHX). The study also investigated in vitro virulence factors, including biofilm formation ability, cell surface hydrophobicity (CSH) and β-hemolysis, to explore aspects of pathogenesis. In our study, regardless of the isolation source, VAN-resistant (VAN-R) and CIP-resistant (CIP-R) <i>E. faecalis</i> and <i>E. faecium</i> were detected. The highest proportion of CIP-R strains was found among clinical isolates of <i>E. faecalis</i> and <i>E. faecium</i>, with clinical <i>E. faecium</i> also showing the highest proportion of VAN-R strains. But the highest proportion of VAN-R <i>E. faecalis</i> strains was found in wastewater samples. The highest TCL MIC₉₀ values for <i>E. faecalis</i> were found in wastewater isolates, while for <i>E. faecium,</i> the highest TCL MIC₉₀ values were observed in food isolates. The highest CHX MIC₉₀ values for both <i>E. faecalis</i> and <i>E. faecium</i> were identified in clinical specimens. The results obtained for <i>E. faecalis</i> did not indicate differences in TCL MIC and CHX MIC values with respect to sensitivity to VAN and CIP. Higher CHX MIC₅₀ and CHX MIC₉₀ values were obtained for CIP-R and VAN-R <i>E. faecium</i>. Among the tested isolates, 97.75% of the <i>E. faecalis</i> isolates produced biofilm, while 72.22% of the <i>E. faecium</i> isolates did so as well. In biofilm-forming strength categories III and IV, statistically significantly higher proportions of CIP-susceptible (CIP-S) and VAN-susceptible (VAN-S) <i>E. faecalis</i> were determined. In category III, there is no statistically significant difference in <i>E. faecium</i> CIP sensitivity. In category IV, we had a significantly higher proportion of CIP-R strains. On the other hand, the association between the moderate or strong category of biofilm formation and <i>E. faecium</i> VAN susceptibility was not significant. <i>E. faecalis</i> isolated from wastewater had a CSH index (HI) ≥ 50%, categorizing them as “moderate”, while all the other strains were categorized as “low” based on the CSH index. Among the <i>E. faecalis</i> isolates, cell surface hydrophobicity indices differed significantly across isolation sources. In contrast, <i>E. faecium</i> isolates showed similar hydrophobicity indices across isolation sources, with no significant difference found. Moreover, no correlation was found between the enterococcal cell surface hydrophobicity and biofilm formation in vitro. After anaerobic incubation, β-hemolytic activity was confirmed in 19.10% of the <i>E. faecalis</i> and 3.33% of the <i>E. faecium</i> strains.