The objective of this study was to investigate the potential use of bacteriophage in preventing E. coli mastitis on dairies. A cocktail consisting of four distinct bacteriophages was generated by screening against 36 E. coli isolates from dairy cows in Washington State with clinical mastitis. The bacteriophage significantly inhibited growth of 58% of the Washington State isolates and 54% of E. coli mastitis isolates from New York State, suggesting that the cocktail of phages had a broad spectrum of action against relevant strains from two distinct geographies. For those E. coli that were completely inhibited by the phage cocktail, an MOI as low as 10 had the same effect as 10 µg/ml of ceftiofur on the growth rate of E. coli over a twelve hour period using optical density measurements. Previous work with Staphylococcus aureus and phage K has shown that bacteriophage activity is inhibited in raw milk; however a 3.3-5.6 log reduction of E. coli growth was achieved when co-incubated with our phage cocktail in raw milk over a twelve hour period at physiologic temperature. A modified gentamicin protection assay using bovine mammary epithelial cells provided a model to test whether bacteriophage could prevent cell attachment and invasion by chronic coliform mastitis strains. Pre-treatment of cell cultures with the phage cocktail significantly reduced adhesion and intracellular survival of E. coli as compared to controls. When combined with a bismuth-based teat sealant, the phage cocktail was able to inhibit bacterial growth when challenged with 1.6 x 103 CFU/mL of a mastitis E. coli strain. Results in vitro suggest that a bacteriophage-based dry cow treatment could reduce the incidence of coliform mastitis in dairy cattle; however it must first be shown that a specific dose of bacteriophage can protect cows from experimentally induced E. coli mastitis without inducing an inflammatory reaction.