The current investigation is focused on the antibacterial potential of Withania somnifera Dunal roots. Ashwagandha root powder contains many bioactive components like alkaloids, tannins, flavonoids, and phenolic compounds. These substances have a variety of recognised medicinal, antimicrobial, and antibacterial properties. This study has investigated the antibacterial activity of W. somniferaroot ethanol extract (Ws-REE). Three concentrations, 0.5, 1, and 2%, were employed for the treatment. The concentration of E. Coli cell culture was determined by using a spectrophotometer. The control group exhibited the highest measured cell density of 1.3 X 109cells/mL. In the measured concentration, the dose-dependent impact was noted. For 0.5, 1, and 2% of the sample, the bacterial cell density was 4.24 X 108, 1.2 X 108, and 8.0 X 106, respectively. The growth inhibitory activities of W. somnifera extract against E. coli may be due to specific chemical compounds or synergistic effects of the compounds present in the extract.