The present study was carried out to evaluate the antibacterial activity and to compare the potency of the drug Majoon-e-Maddat- ul-Hayat Jadwari against the uropathogenic strains of Escherichia coli isolated from the urine samples of diabetic mellitus patients and pregnant women. Majoon-e-Maddat-ul-Hayat Jadwari, a polyherbal Unani formulation was prepared at laboratory scale and assessed for its potency against the clinical strains of uropathogenic Escherichia coli using the cup plate method. The Minimum Inhibitory Concentration (MIC) of the drug was also determined using agar diffusion method. Different strains exhibited different mode of sensitivity against the drug. On comparison, the E.coli isolated from the urine sample of pregnant women exhibited more sensitiveness to the drug than the E.coli isolated from the urine of diabetic mellitus patients. Some strains of E.coli irrespective of their source, did not exhibit any activity. The MIC of the drug was found to be within the range from 150 mg/ml to 200mg/ml in case of E.coli strains isolated from the urine of diabetes mellitus patients and 75mg/ml to 200mg/ml against the E.coli strains from pregnant women.