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Enterococcus faecalis is a major nosocomial uropathogen. We report here the epidemiological investigation of drug resistance and in vitro biofilm formation among E. faecalis strains isolated from patients with urinary tract infection in the Charles Nicolle Tunisian hospital from October 2015 to March 2016. The microbial isolates obtained after urine cultivation on CLED agar were identified by Gram staining, oxidase or catalase reaction, and conventional biochemical tests. The susceptibility to antibiotics was determined by the disk diffusion method according to recommended French standard. Biofilm formation was quantified using the microtiter plate assay with crystal violet staining. The 28 E. faecalis urinary isolates showed systematic susceptibility to rifampicin, vancomycin, teicoplanin, and linezolid; high rates of resistance (75 to 96.4%) to erythromycin, tetracycline, sulfamethoxazole-trimethoprim; medium rates of resistance to norfloxacin and gentamicin (57.1%); and low resistance frequencies (3.6 to 7.1%) to ampicillin, imipenem, and chloramphenicol. Multi-resistance to at least three different classes of antibiotics was detected in 20 isolates (71.4%). The patients infected by E. faecalis were predominantly men with a mean age of 47 years, with an underlying disease. All the 28 E. faecalis isolates effectively formed biofilms to varying degrees. The correlation between biofilm formation, multi-resistance to antibiotics, and underlying diseases is discussed.