The aim of this study was the phenotypic and genotypic analysis of the antibiotic resistance and virulence markers in enterobacterial strains isolated from the hospital environment. In this purpose, 100 enterobacterial strains isolated from hospital surfaces were investigated for their susceptibility patterns, for the ability to colonize the cellular (HeLa) and inert substrate and for the production of soluble, enzymatic factors. The bacterial strains were also investigated for the presence of resistance and virulence genes (aggR, aggA, EaggEC, EAST1, hlyA). The enterobacterial strains isolated from the hospital surfaces exhibited high levels resistance rates to beta-lactams, including 3'd generation cephalosporins, teteracyclines, sulphametoxazole and nalidixic acid. The PCR analysis demonstrated the presence of TEM1, tetA, tetB, tetC, dfrA12, sulI and sulII in a low percentage of the resistant strains. The majority of the tested strains exhibited ability to colonize the inert and cellular substrate and also the ability to produce a series of soluble enzymes implicated in enteric and extra-intestinal pathogenesis (pore forming enzymes, proteases, mucinases, iron chelating agents). The presence of beta-haemolysis on sheep blood agar was well correlated with the presence of hlyA gene, while the aggregative adherence pattern with the presence of aggA, aggR and EAST/1 genes, in different combinations. Our results are demonstrating that the E. coli strains isolated from the hospital environment harbor phenotypic and genetic virulence markers, thus contributing to the development of resistance and virulence genes reservoirs with potential implication for the human health in the hospital environment.