Enterococcus faecium, especially those showing multidrug resistance, has emerged as a significant cause of healthcare-associated infections worldwide. However, relatively little is known about the virulence and pathogenesis of this species. The aim of this study was to determine the occurrence of four putative virulence determinants of E. faecium and to correlate them with phenotypic traits. Using forty E. faecium vanA-type isolates from hospitalized patients and their environmental vicinity, we determined the following: the antimicrobial susceptibility profile, occurrence of the genes cylA, efaA, esp, and gelE, hemolytic and gelatinase activities, capacity to form biofilm and in vitro adhesion to epithelial cells. All isolates were shown to be resistant to vancomycin and teicoplanin, as well as to two or more other antimicrobials. All isolates harbored at least one putative virulence marker, and the prevalence was as follows: esp, 87.5%; efaA, 82.5%; gelE, 70%; and cylA, 65%. The presence of 4 genes was observed in 32.5% isolates. The presence of the efaA was associated with the presence of esp, regardless of the source of the isolates. A positive association with the presence of cylA and hemolytic activity in the sheep blood agar assay was observed. No association was found for gelE and gelatinase production in the agar plate assay, for efaA and LLC-MK2 cell adhesion, and for esp and biofilm formation on polystyrene surface. These results show the presence of putative virulence genes in multiple antimicrobial resistant E. faecium isolates from different sources in a hospital setting.