The present study was undertaken to evaluate the mode of antibacterial activity of Eclalbasaponin isolated from Eclipta alba, against selected Gram-positive and Gram-negative bacteria. The probable chemical structure was determined by using various spectroscopic techniques such as Fourier transform infrared spectroscopy (FTIR) and mass spectroscopy. The antibacterial activity was evaluated by well diffusion technique, pH sensitivity, chemotaxis, and crystal violet assays. Eclalbasaponin showed clear zone of inhibition against both Bacillus subtilis and Pseudomonas aeruginosa and exhibited growth inhibition at the pH range of 5.5-9.0. The isolated saponin exhibited its positive chemoattractant property for both bacterial strains. Results of crystal violet assay and the presence of UV-sensitive materials in the cell-free supernatant confirmed the cellular damages caused by the treatment of Eclalbasaponin. The release of intracellular proteins due to the membrane damage was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Changes in the cell surface structure and membrane disruption were further revealed by FTIR and scanning electron microscopy analysis. The present study suggests that the isolated saponin from E. alba causes the disruption of the bacterial cell membrane which leads to the loss of bacterial cell viability.