The diversity in structure and herbicidal properties detected in natural phytotoxic compounds could bring about advantages for development bio-herbicides. The present study was carried out search for potential weed inhibitors from the parts of Mimosa pigra L. The ethyl acetate (EtOAc) extract of leaf of M. pigra showed inhibitory activity during the time that Echinochloa crus-galli (barnyardgrass) germinates and grows, which is greater than that of other extracts. From this active extract, potent growth inhibitors were isolated and identified by column chromatography (CC), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (1H and 13C NMR). The six compounds were purified in this study namely: lupeol (C1, 13.2 mg), stigmastane-3,6-dione (C2, 14.7 mg), quercetin (C3, 20.2 mg), chrysoeriol (C4, 28 mg), methyl gallate (C5, 21.5 mg) and daucosterol (C6, 16.0 mg). The C2 (quercetin) compound completely inhibited the emergency, shoot height and root length of E. crus-galli at 1 mg/mL concentration (IC50 shoot height =0.56 mg/mL). This was also the first study to report the isolation and allelopathic activity of lupeol, chrysoeriol and daucosterol from M. pigra leaf. Findings of this study highlighted that quercetin from M. pigra may become bio-herbicide to control barnyard grass and other grass weeds for the development of safe agriculture.