Multi-drug resistance in clinical isolates has become a serious problem due to a progressive decline in the number of antibiotics effective for treatment of human infections. The connection between the antibiotic resistance of food animal origin and that of clinical isolates and community health has been suggested, however, this remains a controversial issue because the ecology of antibiotic resistance is not well understood. Insects such as house flies that develop in decaying organic substrates including, animal manure/feces, may disseminate antibiotic resistant bacteria in the rural and urban environment. House flies and animal manure were collected from a cattle feedlot (where antibiotic are heavily used growth promotion, prophylaxis, and treatment) and from the American bison (with no antibiotic use) and screened for enterococci. Enterococci were quantified, identified, and characterized for antibiotic resistance and virulence factors and mobile genetic elements by polyphasic approach (phenotype and genotype). 223 isolates (house flies) and 174 (manure) were isolated and characterized form the cattle feedlot; 141 isolates (house flies) and 290 isolates (manure) were identified and characterized from the bison environment. Our results demonstrate that antibiotic resistance is more prevalent in the confined cattle environment (manure and flies). The isolates represented E. faecalis, E. faecium, E. gallinarum, and E. casseliflavus and commonly carried resistance genes (tetS, tetM, tetO, ermB) and conjugative transposon Tn916. This study shows that antibiotic resistant strains are common in the confined cattle environment and house flies likely play an important role in the ecology of these bacteria in the agricultural environment.