Abstract: Background: In recent years, drug resistance to human pathogenic bacteria has been commonly reported from all over the world. The use of plant compounds to treat infections is an age-old practice in developing countries, where there is dependence on traditional medicine for a variety of diseases. Interest in plants with antimicrobial properties has revived as a result of current problems associated with the use of antibiotics. Materials and Methods: This study is designed to isolate phytoconstituent 3, 4, 4a, 5, 8, 8a-hexahydro-6-methylisochromen-1-one (HMIC) from leaves extract of Stachytarpheta cayennensis and test the antibacterial activity against different pathogenic bacterial species and in silico glucosamine-6-phosphate synthase (GlcN-6-P) inhibition property of the HMIC. The phytoconstituents HMIC was isolated from the crude ethanolic extract and purification was carried out by column chromatography using silica gel (100-200 mesh size) and n-hexane-ethyl acetate (7:3) as eluting system, the compound was characterized by analytical 1HNMR, 13C NMR, IR and mass spectral data. The antibacterial activity of HMIC was evaluated against Gram-positive and Gram-negative bacteria using the agar-well diffusion method and automated docking was used to determine the orientation of inhibitors bound in the active site of GlcN-6-P synthase employing AutoDock 3.0. Results: The phytoconstituent HMIC showed the strongest antibacterial activity against Klebsiella pneumoniae followed by Pseudomonas aeruginosa where as it showed moderate activity on Staphyllococcus aureus of the bacterial growth. It also possesses better glucosamine-6-phosphate synthase inhibition in molecular docking studies with minimum docking and binding energy and better ligand efficiency when compared to standard. Conclusion: This compound was isolated for the first time from this plant and no evidence could be found for the previous reported presence of HMIC in the genus Stachytarpheta. Considering the antibacterial activity, this could offer a scientific basis for the therapeutic potency of Stachytarpheta cayennensis used in traditional medicine. Further studies are necessary to determine the toxicity, side effects, circulating levels, pharmacokinetic properties, diffusion in different body sites and the mechanism involved with the antibacterial activity of HMIC.