Objective: The objective of the study was to evaluate the antibacterial activity of methanolic stem fraction of Tinospora cordifolia against Escherichia coli and Staphylococcus aureus by in vitro and in silico approaches. Methods: In agar disc diffusion method, the inhibitory zone produced by various concentrations of the fraction showed a dose-dependent inhibition pattern. Minimum inhibitory concentration (MIC) values were calculated by broth dilution method. The total DNA present in the fraction treated bacterial cultures was estimated and compared with control DNA. The two-dimensional and three-dimensional structures of the gas chromatography– mass spectrometry (GC–MS) identified compounds were generated using ChemSketch tool. The docking studies were performed for analyzing the receptor and ligand interactions. Results: The higher zone revealed the maximum inhibition of the growth of bacteria that were ranged from 2 mm to 6 mm for E. coli and 1.5 mm to 6.3±0.29 mm for S. aureus. MIC values showed that 30 μg/ml of the fraction was found as the effective dose. The DNA content isolated from the treated culture of both the strains was comparatively lesser than that of the untreated control culture. The GC–MS data analysis depicted the presence 15 major components in the fraction and the sharp peaks were obtained at time intervals 17.50, 20.27, 30.06, etc. Conclusion: Thus, methanolic stem fraction of T. cordifolia possesses promising therapeutic activity against the urinary tract infection pathogens such as E. coli and S. aureus and a further exploration in the isolation and characterization such as plant-derived phytoconstituents would open up new ventures in the field of antibacterial drug discovery.