In this study, a herbal preparation containing Dalbergia sissoo and Datura stramoium with cow urine (DSDS), was evaluated for its antibacterial potential against pathogenic strains of gram-positive (Staphylococcus aureus and Streptococcus pneumoniae) and gram-negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) bacteria. Antibacterial activity was compared to standard antibiotic drugs i.e. Chloramphenicol (30 mcg), Ampicillin (10 mcg), Nalidixic acid (10 mcg) and Rifampicin (30 mcg). Cow urine extract was found to be most active against both gram-positive as well as gram-negative bacteria. Clinical isolate of S. aureus showed higher sensitivity towards cow urine extract of DSDS than standard strains, and inhibited growth on most regulatory levels such as inhibition of protein, DNA, RNA and peptidoglycan synthesis. The results of the present study shows that the cow urine extract of DSDS may be used as a potent antiseptic preparation for prevention and treatment of chronic bacterial infections.