ABSTRACT: Cow urine therapy and all traditional practices from Indian systems of medicine have a strong scientific base. The cow has proved to be a boon in the areas of agriculture, science and technology, industry, energy, medicine etc for the development of any nation, in addition being eco-friendly in nature. In the present study the antibacterial potentials of cow urine were investigated. Total 14 pathogenic and non-pathogenic bacterial cultures were used as test organism against 10 different cow urine samples. The highest zone of inhibition was shown by sample G against P aeruginosa NCIM 2945 (1.8cm) while the smallest zone of inhibition was shown against E. coli NCIM 2065(0.3 cm) by sample A. Based on cumulative effect against the test organism, the urine sample G was found to be the most efficient inhibiting all the 14 test cultures. The antibacterial activity reported by sample G was comparable with standard antibiotics. A higher zone of inhibition was observed by sample G against P aeruginosa NCIM 2945 as compared to that of Gentamicin, Oxacillin and vancomycin. Though the urine sample G showed a strong antibacterial activity against all the test organisms, but the activity was reported low against the entire Gram positive bacteria compared to Gram negative bacteria. The presence of proline which is considered as a major amino acid in antimicrobial peptides was also observed in the urine sample G.