Initially commensal bacteria of the gastrointestinal tract, Enterococcus species are opportunistic pathogens that can colonize the female vaginal tract, particularly in patients with aerobic vaginitis or after receiving antibiotic treatment. Enterococcus spp., are associated with a wide range of illnesses, particularly in patients with altered host microbiota or immunocompromised conditions.c. Here we assessed the antibiotic susceptibility, the biofilm formation ability and the production of hemolysin and gelatinase in enterococcal isolates from vaginal microbiota with phenotypic characterization methods. Our results revealed the prevalence of E. faecalis strains from vaginal samples. 12.9% of E. faecalis were resistant to glycopeptides while E. faecium isolates showed multi-resistant profile to ?-lactamine antibiotics, aminoglycosides and macrolides. Biofilm was detected in thirty isolates, of which four were strongly biofilm-producing and thirteen moderately biofilm-producing. Moreover, hemolysin and gelatinase virulence factors were detected in 83.9% and 89.3% of Enterococcus spp strains respectively. In addition to biofilm formation, our results demonstrated that hemolysin and gelatinase production are significant virulence factors in Enterococcus spp isolated from vaginal microbiota.