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Background: Owing to restricted treatment options, Vancomycin-resistant Enterococci (VRE) was considered a prominent cause of nosocomial infections. This study was undertaken to evaluate the presence of Van-type and virulence determinants in the clinical isolates of E. faecium (Ent. faecium) in Shariati Hospital. Materials and Methods: A total of 150 Enterococcal isolates were surveyed. Antimicrobial susceptibility testing was performed by disc diffusion and E-test as well as the genotypic method. The presence of virulence factors, including hyaluronidase (hyl), gelatinase (gelE), aggregation substance (asa1), and Enterococci surface protein (ESP) were identified by Polymerase chain reaction (PCR). Results: Overall, 66.67 percent (80/120) of VRE Ent. faecium strains were confirmed by the PCR method. The maximum number of isolates was from urine specimens (p < 0.05) and blood samples. Among the 80 VRE Ent. faecium isolates, 76 isolates showed high-level resistance (MICs to Vancomycin 32 > µg/ml) and carried a VanA phenotype (p < 0.05). In all the isolates, asa1, gelE, and ESP genes were identified in 14% (17/5), 26/3% (21/80), and 45% (36/80), respectively. E. Ent. faecium carried ESP at a significantly higher frequency presented in VRE strains (p < 0.001). The prevalence of hly determinants in the E. faecium was 20% (16) (P < 0.001). Conclusion: We, in our hospital, are faced with a high rate of VRE Ent. faecium isolates with a VanA-positive phenotype. With increasing resistance of the VRE strains to linezolid, we will encounter a serious challenge in treating VRE patients in future years. An interesting finding from the present study is that the spreading rates of ESP and hly among Ent. faecium isolates are higher.