Enterococcus faecium has become an increasingly important nosocomial pathogen due to formation of biofilms on several surfaces. Sixty one (61) E. faecium strains isolated from blood, urine and fecal were assessed for biofilm production, the effect of different glucose concentration on biofilm production and also the presence of esp, fsr and gelE genes. Pulsed field gel electrophoresis (PFGE) method was performed to show chromosomal similarities and also to determine correlation between biofilm formation ability and genetic identity of E. faecium strains. It was observed that glucose concentration of the medium and incubation period can affect biofilm formation of the bacteria. When tested strains were incubated in a medium containing 1% glucose for 48 h, 66.66% of urine isolates, 60.71% fecal isolates and 25% of blood isolates produced strong biofilm structures. esp-positive strains (80% of the all isolates) were also identified as strong biofilm producers compared to esp-negative isolates. As a result of PFGE analyses, isolates numbered 14 (isolated from fecal sample) and 81 (isolated from blood sample) were classified in minor group B at a level of 48% similarity. Out of these two isolates, all the isolates were included in major group A with 43% similarity level and this group was subdivided into six subgroups.