Flacourtia indica (Burm. F). Merr., a plant widely used in ethnomedicine, was investigated for its antibacterial properties and chemical composition using preparative thin-layer chromatography (TLC) bioautography, gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) spectroscopy. Bioautography was performed on silica gel-coated TLC plates against Streptococcus pneumoniae, Vibrio cholerae, and Escherichia coli. The ethanolic extract exhibited a clear inhibition zone against E. coli, while the chloroform extract showed significant inhibition against S. pneumoniae. GC-MS analysis revealed 48 compounds in the ethanolic extract and 20 in the chloroform extract. Major compounds identified in the chloroform fraction included sulfurous acid hexyl nonyl ester, phenol derivatives, phthalic acid esters, siloxanes, heneicosane, and piperidinone derivatives. To further confirm the bioactive constituents, 1H NMR spectroscopy was performed on bands exhibiting antibacterial activity. The analysis indicated the presence of aliphatic acyclic, β-substituted aliphatic, and α-monosubstituted aliphatic compounds. These findings suggest that the chloroform extract of F. indica contains several aliphatic group chemicals with notable antibacterial activity. The study highlights the potential of isolating, purifying, and characterizing these compounds for pharmaceutical applications, reinforcing the ethnomedicinal relevance of F. indica as a source of bioactive molecules.