Cow urine has tremendous applications in agriculture. It is shown to possess inhibitory activity against many phytopathogenic fungi and bacteria. The present study was conducted to determine antibacterial and antifungal efficacy of cow urine. The well-plate assay showed marked growth of inhibition against Aspergilus niger, Fusarium oxysporum, Xanthomonas auxonopodis,Psedomonas solancearum, Xanthomonas cucurbitae by cow urine as well as microbs isolated from cow urine. The TLC and titrimetric analysis were showed presence of lipase enzyme in the cow urine. Moreover, sterilized cow urine can be utilized as natural growth medium for Pseudomonas fluorescence, Bacillus subtilis, mixture of fungi and yeast. The use of cow urine can be the cost-effective and eco-friendly approach for controlling various diseases of plants.