Background: Enterococci possess many virulence genes implicated in their pathogenesis. Biofilm help the organism to colonize and cause infections. Objectives: This study was set out to investigate and compare biofilm formation ability and presence of asa1 and esp genes among E. faecium and E. faecalis isolates from diverse sources. Furthermore, association between biofilm formation, esp, asa1 genes and vancomycin resistance was analyzed. Methodology: 76 pathogenic enterococcal isolates and 36 enterococcal isolates from healthy individuals were collected. All isolates were investigated for biofilm using microtitre plate, asa1and esp genes were detected by primer-specific PCR, vancomycin resistance were screened using agar method and confirmed by PCR. Results: the majority of clinical isolates (80.5%) were biofilm producers, however biofilm was detected only in 36.4% of colonizing isolates. E. faecalis (82.6%) produced biofilm more than E. faecium (36.1%), esp gene (48.5%) was presented more than asa1gene (15.2%). Virulence genes were detected in high rates among biofilm producers isolates, low vancomycin resistance rate was seen among isolates which produced slim layers. Conclusion: biofilm was detected in high rate in E. faecalis harbored esp gene. Biofilm, asa1 and esp genes were more presented among isolates from non invasive sites than invasive sites, thus aids enterococci to provoke clinical infections, lower biofilm was seen in E.faecum. However, vancomycin resistant isolates produced less slim than vancomycin sensitive.