Mastitis, a disease with high incidence worldwide, is the most prevalent and costly disease in the dairy industry. Environmental mastitis pathogens, such as Escherichia coli (E. coli), are major etiological agents of bovine mastitis in well-managed dairy farms. However, there is still a need to develop more efficient, safe, and economical treatments for mastitis. In the current research, Heter-LP, a new system biology-based method of drug repositioning, was applied to potentially identify novel therapeutic avenues for the treatment of E. coli mastitis. On-line data repositories relevant to known diseases, drugs, and gene targets along with other specialized biological information for E. coli mastitis, including key genes with robust bio-signatures, drugs and related diseases were used as input data for analysis with the Heter-LP algorithm. Our analyses identified novel drugs such as Glibenclamide, Ipratropium, Salbutamol, and Carbidopa as possible therapeutics that could be used against E. coli mastitis. Predicted relationships can be used by pharmaceutical scientists or veterinarians to find commercially efficacious medicines or combination of two or more active compounds to treat mastitis.