House flies (HF) develop in decaying organic substrates (e.g. animal manure) and may disseminate antibiotic resistant strains in the environment. Due to an extensive use of antibiotics in clinical medicine and animal productions, the development and spread of the antibiotic resistant bacteria have become a serious problem. The connection between the antibiotic resistance of the food animal origin and that of clinical isolates has been suggested. In this study, HF and animal manure were collected from a cattle feedlot (frequent use of antibiotics) and from the American bison (no antibiotic use) and screened for enterococci. Enterococci were cultured, quantified, identified, and characterized for antibiotic resistance and virulence factors and mobile genetic elements by a polyphasic approach. Our results demonstrated that antibiotic (mainly tetracycline and erythromycin) resistant enterococci were more prevalent in manure and flies from the confined cattle environment than that of the bison environment. The isolates were represented by E. faecalis, E. faecium, E. gallinarum, E. casseliflavus, and E. hirae and commonly carried resistance genes (tetS, tetM, tetO, ermB). The dominant species in a feedlot was E. hirae (92.0%) in manure and E. faecalis (38.5%) in flies. In the bison environment, E. casseliflavus (90.4%) was dominant in manure and E. faecalis (57.6%) in flies. Moreover, feedlot HF showed significantly higher prevalence of clinically important isolates such as E. faecalis with resistance genes than flies from the bison site. This study shows that HF likely play an important role in the ecology of antibiotic resistant bacteria in the agricultural environment.