Enterococcus faecalis ranks as one of the leading causes of nosocomial infections including urinary tract infections, surgical wound infections and endocarditic. Epidemiological links have been reported between E. faecalis inhabiting non-human sources and environmental sources. This study investigates the genetic diversity, antibiotic resistance and virulence determinants in E. faecalis from chicken and cattle feces, river water, sewage wastewater, farm animal drinking water and urinary tract infected patients in Malaysia. Sampled were obtained at two different sampling times, June and December 2014. A total of 250 E. faecalis isolates were obtained consisting of 120 isolates from farm animals, 100 isolates from water sources and 30 isolates from hospitalized patients. Pulse-field gel electrophoresis-typing yielded 63 pulsotypes, with high diversity observed in all sources (D=≥0.901). No pulsotype was common to the three sources. Each patient room had its own unique PFGE pattern which persisted after six months. Minimum inhibitory concentrations (MIC) of Vancomycin, Gentamicin, Penicillin, Tetracycline, Nitrofurantoin, Levofloxacin, Ciprofloxacin and Fosfomycin were evaluated. Resistance to Tetracycline was most prevalent in isolates from farm animals (62%) and water sources (49%). Water isolates (86%) had a higher prevalence of the asa1 gene, which encodes for aggregation substance, whereas clinical (78%) and farm animal isolates (87%) had a higher prevalence of the esp gene, encoding a surface exposed protein. This study generates knowledge on the genetic diversity of E. faecalis with antibiotic resistance and virulence characteristics from various sources in Malaysia.