Aim: 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 Rf 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.