From the ancient period cow's urine has been used as a medicine. In Veda, cow's urine was compared to the nectar. In Susrut, several medicinal properties of cow's urine have been mentioned and are known to cause weight loss, reversal of certain cardiac and kidney problems, indigestion, stomach ache, edema, etc. However, the literature and scripture did not mention the antigenotoxic properties of cow's urine. In the present investigation, the antigenotoxic/antioxidant properties of cow's urine distillate and redistillate were studied in vitro. The antioxidant status and volatile fatty acid levels were determined. Actinomycin-D (0.1 micromol/L) and hydrogen peroxide (150 micromol/L) were used for inducing DNA strand break with 0.1% DMSO as negative control. Dose for the antigenotoxic effect of cow's urine was chosen from the dose response study carried out earlier. Both actinomycin-D and H202 caused statistically significant DNA unwinding of 80% & 75% respectively (P < 0.001) as revealed by fluorimetric analysis of DNA unwinding (FADU), and the damage could be protected with the redistilled cow's urine distillate (1, 50 & 100 microL) in simultaneous treatment with genotoxic chemicals. The redistillate of cow's urine was found to possess total antioxidant status of around 2.6 mmol, contributed mainly by volatile fatty acids (1500 mg/L) as revealed by the GC-MS studies. These fatty acids and other antioxidants might cause the observed protective effects.