Background and objectives: The chemical composition of Melanthera scandens (MS) methanol (MeOH) extract is yet to be fully comprehended. Chemical composition of a plant extract is closely related to the biological activity of the plant material, in phytomedicine. In this study, chemical analyses of MS extracts were carried out with the aim of identifying the organic chemical constituents. Methods: Powdered MS leaf was soxhlet extracted with dichloromethane and MeOH. The solvent extracts were analyzed using electrospray ionization-mass spectrometer and GC-MS (fatty acid methyl ester and trimethylsilyl derivatives). Fresh MS leaves were cold extracted with MeOH for biological studies and isolation of compounds. The MeOH extract was screened for phytochemical constituents and partitioned with n-hexane, ethyl acetate (EtOAc) and n-butanol to give the respective fractions. The n-hexane fraction was examined for volatile constituents using gas chromatography-mass spectrometry. Antibacterial study of n-hexane, EtOAc, butanol fractions and crude MeOH extract was undertaken; extract and fractions were screened against E. coli, P. aeruginosa, B. subtilis, B. cerus and S. aureus. The minimum inhibitory concentration and minimum bactericidal concentration of the EtOAc fraction (the most active) were determined using the macrobroth dilution method, and the fraction was subjected to column fractionation. Eluents were monitored by thin-layer chromatography. The combined fractions that exhibited antibacterial activity were further purified to obtain isolates which were characterized by spectroscopic techniques-nuclear magnetic resonance, Fourier transform infrared, gas chromatography-mass spectrometry (fatty acid methyl ester and trimethylsilyl) and electrospray ionization-mass spectrometer. Results: Phytochemical screening of MS extracts showed the presence of alkaloids, tannins, cardiac glycoside, saponins and steroid, while flavonoid was not detected. The n-hexane fraction, EtOAc fraction, and crude MeOH extract inhibited the growth of Bacillus species, while the butanol fraction did not display any activity against the tested microbes. The EtOAc fraction showed the greatest activity. Gas chromatography-mass spectrometry analysis of the n-hexane fraction identified 14 compounds, among which phytol was of highest percentage composition. Ten different compounds were identified in the dichloromethane extract, consisting mainly of oleanane and ursolane derivatives. Electrospray ionization-mass spectrom