Background: The role of the hospital environment as a source of pathogenic bacteria in recent studies has been poorly investigated. This study investigated the distribution of antimicrobial resistance genes and virulence determinants in Enterococcus species isolated from hospital environment in Sari, Iran. Method: Overall, 90 enterococci strains were obtained from high touch surfaces of four hospitals in Sari, Iran. These environmental samples were obtained from bathroom, beds, tables, doorknobs, room keys, wheelchair and walls in the patient and staff’s rooms. The resistance profile of the isolates was determined by disk diffusion method. Seven resistance genes and two virulence associated genes were evaluated molecularly by multiplex PCR. Results: According to the PCR, 42 (46.66%) of them were E. faecalis and 48 (53.33%) others were detected as E. faecium. Also, 28 (66.6%) E. faecalis and 18 (37.5%) E. faecium isolates were multidrug-resistant (MDR). Among all 90 environmental isolates 54 (60%), 54 (60%), 8 (8.8%), 8 (8.8%), 60 (66.6%), 26 (28.8%), and 24 (26.6%) isolates contained tetM, tetL, vanA, vanB, ermB, aac(6´)-Ie-aph(2´´)-Ia, and aph (3´)-IIIa, respectively. Moreover, all isolates were investigated for the presence of virulence genes and 88 (97.7%) of isolates had esp gene, and 16 (17.7%) had ace.Conclusions: This report showed that the environmental isolates of Enterococcus are the major sources of antibiotic resistance genes that can transfer them to the clinical isolates of bacteria in hospital settings. An effective following strategy should be organized to clearance and stop emergence of these pathogenic bacteria.