The present study mainly focused the antibacterial activity of cow urine with three formulations, fresh cow urine, photo activated cow urine and neem oil with photo activated cow urine were screened for antibacterial activity against bacterial pathogens (E. coli, B. subtilis, P. aeroginosa, K, pneumonia, S. aureus and S. pyogenes) by using well diffusion method. Fresh cow urine shown effective antibacterial activity than photo activated cow urine and neem oil photo treated cow urine. The maximum growth inhibition of cow urine shown against P. aeroginosa (18.0±0.5 mm) followed by S. aureus (17.5±0.5 mm) and K. pneumonia (17.5±0.5 mm). The neem oil with photo activated cow urine has maximum growth inhibition against P. aeroginosa and S. aureus (16.1±0.2mm) respectively. However the least growth inhibition observed in photo activated cow urine against B. subtilis (10.9±0.3 mm). These results are indicated that the presence of certain volatile and phenolic compounds in fresh cow urine, when compare with photo and neem oil treated.