Abstract: Herbs are largely unexplored source of drug repository. Medicinal plants have great potential for providing novel drug leads with novel mechanism of action. The present study describes the antibacterial activity, phytochemical profile and toxicological evaluation of Urtica dioica. U. dioica leaves were subjected to solvent extraction by hexane, ethyl acetate, chloroform, methanol and water. All the extracts were tested for antibacterial activity against five clinical isolates of Gram positive and Gram negative bacteria by disc diffusion method. Hexane extract showed good antibacterial activity against all the five bacterial strains, hence it was further purified using silica column chromatography and Minimum Inhibitory Concentration (MIC) of the semi purified fraction-B (FB) of hexane extract was determined by serial tube dilution method. FB showed MIC value of 31.25, 250, 7.81, 31.25 and 125 μg mL-1 against Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumoniae and Shigella flexneri, respectively. FB was subjected to GC-MS analysis in search of potent antibacterial compound(s). Neophytadiene (26.97%) butyl tetradecyl ester (9.53%), Dibutyl phthalate (7.45%), Bis (2-ethyl hexyl) maleate (8.80%) and 1,2-benzenedicarboxylic acid (9.89%) were the major constituents which may be responsible for the antibacterial activity of FB of U. dioica. Sub-acute oral toxicity of FB was carried out in wistar rats at doses of 100, 200, 400 and 800 mg kg-1 Body Weight (b.wt.) to assess index of safety. FB showed MIC value as low as 7.81 μg mL-1 against Salmonella typhi, exhibiting that a promising antibacterial agent is present in this fraction. Hematological and biochemical parameters showed that the FB was safe at tested concentrations. Hence further purification of FB is required to obtained potential antibacterial compound.