Introduction and Aim: In Ayurveda, cow's urine has been reported to have many therapeutic values. The raising trend in antibacterial resistance is a cause of concern and demands an alternative therapeutic agent. Hence, the present study aims to know the antibacterial properties of urine from cows on clinical and reference strains of bacteria.     Materials and Methods: The present in vitro experimental study, used agar dilution method to know the Minimum Inhibitory Concentration (MIC) of cow’s urine distillate (CUD) on few reference bacterial strains and Enterococcus faecalis, Staphylococcus aureus, Salmonella Typhi, Escherichia coli, and S. Paratyphi A isolated from clinical samples. Resistance and sensitivity of these isolates to routinely used antibiotics was studied by Kirby Bauer’s disk diffusion method.     Results: MIC of CUD for different standard bacterial strains varied from 250 to 500?l/ml. Among the clinical isolates, all Salmonella Typhi, 85% of S. Paratyphi A, 60% of E. coli, 80% of the S. aureus, 64.28% S. aureus were resistant to methicillin (MRSA) and 24% of E. faecalis were killed by 500?l/ml of CUD. Clinical strains of bacteria, susceptible to routinely used antibiotics, were also found to be susceptible to CUD and showed a concentration-dependent inhibitory effect. Multi drug resistant strains of nine MRSA, one E. faecalis and two E.coli were also susceptible to 500?l/ml concentration of CUD.   Conclusion: CUD can be considered as an alternative anti-bacterial agent for multi-drug resistant bacterial pathogens as it showed concentration dependent inhibitory effect on both Gram-positive and Gram-negative pathogenic bacteria.