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Objective:To evaluate the relationship between biofilm formation and incidence of virulence determinants in clinical isolates of Enterococcus.Methods: In this cross-sectional study, the clinical isolates of Enterococcus strains were collected from the university teaching hospitals in Ahvaz, Iran from June 2017 to June 2018. Then, the prevalence of Enterococcus species, antibiotic resistance, virulence factors, and biofilm-producing ability were determined. Results: Of the 119 tested isolates, 17 (14.3％) were Enterococcus faecalis, 72 (60.5％) were Enterococcus faecium and 30 (25.2％) were other Enterococcus spp. Gelatinase was detected in 97 (81.5％) isolates, enterococcal surface protein in 41 (34.5％) isolates, serine protease in 39 (32.8％) cases, accessory colonization factor in 111 (93.3％) cases and pathogenicity islands in 17 (14.3％) cases. The biofilm formation ability was observed in 75 (63.0％) of all isolates and the association between the presence of enterococcal surface protein gene and biofilm formation was statistically significant. Higher resistance to vancomycin, gentamycin, and teicoplanin was indicated in Enterococcus faecium with 81.8％, 58.4％, and 85.7％ resistance rate, respectively. All Enterococcus faecalis isolates were sensitive to teicoplanin and vancomycin. Conclusions: The presence of antibiotic-resistance with several virulence factors in Enterococcus spp has become a conc. High prevalence of enterococcal surface protein gene among biofilm-producing isolates suggests a potential relation between biofilm formation and the enterococcal surface protein gene, and further studies are needed to identify the mechanism of biofilm inhibition.