The aim of this work was to determine the occurrence of different enterococcal species and the prevalence of antimicrobial resistance and virulence genes in enterococci isolates recovered from faecal samples of 98 healthy human volunteers in Tunisia. Isolates were tested for antibiotic resistance phenotypes, genotypes and virulence genes. In addition, high-level aminoglycoside resistant (HLAR) Enterococcus faecalis and Enterococcus faecium isolates were tested for clonal diversity by pulsed field gel electrophoresis (PFGE). The following species were detected among the 98 enterococci obtained: 51 Enterococcus faecalis, 40 Enterococcus faecium, 4 Enterococcus mundtii and 3 Enterococcus gallinarum. Antibiotic resistance was detected as follows (% in E. faecalis/E. faecium): erythromycin (51 %/55 %), tetracycline (51 %/15 %), pristinamycin (51 %/27.5 %), gentamicin (19.6 %/10 %), kanamycin (25.5 %/12.5 %), streptomycin (29.4 %/7.5 %), chloramphenicol (7.8 %/2.5 %). The tet(M)+/-tet(L) genes were found in 92 % and 83 % of tetracycline-resistant E. faecalis and E. faecium isolates, respectively. High-level resistance for kanamycin and gentamicin was mediated by aph(3')-IIIa and aac(6')-aph(2aEuro(3)) genes. Of erythromycin-resistant enterococci, 85 % harboured the erm(B) gene. The erm(C) gene was found in one erythromycin-resistant E. mundtii isolate. Most high-level gentamicin and/or streptomycin E. faecalis and E. faecium isolates showed unrelated patterns by PFGE. Fifty-two percent of enterococci showed a multidrug-resistant phenotype. The following virulence genes were detected among the 98 enterococci: gelE (13.2 %), esp (20.4 %), cyl (9 %, showing haemolytic activity), ace (48.9 %), and hyl (0 %). In conclusion, E. faecalis and E. faecium are the predominant enterococcal species in the faecal environment of healthy humans, and they present high rates of resistance for antibiotics of clinical relevance, such as aminoglycosides.