The study was aimed to evaluate the pharmacognostic features of Hemidesmus indicus, analyze the antibacterial properties of ethanolic and aqueous root extracts, and identify bioactive compounds using GC-MS analysis. Materials and Methods: We conducted pharmacognostic analyses, including organoleptic, macroscopic, microscopic, and physio-chemical evaluations, phytochemical screening, and fluorescence analysis. We also used standard protocols for TLC fingerprinting. Antibacterial activity against gram-negative bacteria was assessed using the agar well diffusion method. Additionally, we conducted GC-MS analysis to identify major compounds in the plant extracts. Results: In our H. indicus root examination, we identified three regions: wood, inner cortex, and outer phellem. Key findings: moisture content at 9.0% w/w, ethanol and water-soluble extractives at 11.21% and 16.89% w/w, total ash at 4.21% w/w, acid-insoluble ash at 1.14% w/w, and water-soluble ash at 3.12% w/w. Phytochemical screening detected alkaloids, flavonoids, tannins, polyphenols, triterpenoids, carbohydrates, and proteins. TLC analysis of the ethanolic extract showed varying R f values. The aqueous extract exhibited stronger antibacterial activity against E. coli than the ethanolic extract. GC-MS analysis identified over 30 prominent compounds in the ethanolic extract. Conclusion: In conclusion, this study provides valuable insights for the standardization of H. indicus root extracts. The aqueous root extract displayed notable antibacterial properties. Furthermore, our GC-MS analysis identified the presence of phenolic, Fatty acids, and terpenoids compounds in the root extract of H. indicus, which hold potential therapeutic applications.