The indiscriminate use of herbicides can guide to some environmental damages. Thus, the search for alternative bioherbicides from natural sources with biodegradable properties and less toxicity to the microbiota is essential. The focus of this work is the chemistry and phytotoxic study of the hydroethanolic extract of Machaerium amplum Benth. leaves. The chemical study by FIA-ESIIT-MSn experiments allowed the detection of apigenin and luteolin C-glycoside derivatives, quinic acid, the A-type proanthocyanidin dimer and trimer, and a saponin Soyasapogenol B type. Phytotoxicity assays were performed with the extract and a group of fractions against tomato and onion seeds under the parameters of germination, hypocotyl growth, and root length. The extract showed a 70% of inhibition effect at 0.8 mg.mL-1 on tomato root length. For the onion seeds, the percentual of inhibition was 65% at 0.4 mg.mL-1 . All groups of fractions tested showed significant inhibition on tomato seedling root length at 0.8 mg.mL-1 . The flavonoids fraction showed interesting activity at 0.4 mg.mL-1 . The same fraction showed a percentage of inhibition above 50% at 0.2 mg.mL-1 on onion seedling root length. This activity may be associated with the C-flavones presence in this fraction. According to the ecotoxicity test with Artemia salina Leach, the extract presents no toxicity at the concentrations tested, and no antimicrobial activity against Fusarium graminearum, F. oxysporum and Aspergillus flavus, thus reinforcing the potential of the extract.