Background: Enterococci, particularly those that produce biofilm, can cause different infections ranging from simple infections to sepsis. This study aims to understand the effect of sub-inhibitory concentrations of antibiotics (ampicillin, vancomycin, ciprofloxacin and tetracycline) on biofilm formation of Enterococcus and its association with virulence genes (asa1, esp and gel E). Methods: This cross-sectional study included Enterococci strains isolated from different microbiological samples. Disk diffusion and microtiter plate techniques were used to examine the isolates antibiotic sensitivity and biofilm production respectively. Isolates were investigated for asa1, esp and gel E genes using multiplex PCR. Minimum inhibitory concentration (MIC) of ampicillin, vancomycin, ciprofloxacin, and tetracycline for strong biofilm forming isolates was determined by broth microdilution then the sub-MIC effect of these antibiotics on their biofilm formation was evaluated. Results: A total of 80 Enterococcus (65 Enterococcus faecalis and 15 Enterococcus faecium) strains were identified. Compared to Enterococcus faecalis, Enterococcus faecium showed higher antibiotics resistance and lower biofilm formation. asa 1, esp and gel E genes were detected in 44.6%, 60.0% and 76.9% of Enterococcus faecalis and 40.0%, 33.3% and 26.7% of Enterococcus faecium respectively with higher frequency of these genes among biofilm forming isolates. Of the four selected antibiotics, sub-inhibitory concentrations of only ampicillin and vancomycin significantly enhanced biofilm density of Enterococcus faecalis while Enterococcus faecium was not significantly affected. Conclusion: Increased biofilm formation in some isolates at different sub-MIC ensures the significance of appropriate antibiotic use to reduce the danger of resistant bacteria selection.