Plants produce chemical compounds with high nematicidal activity and potential for large-scale application in nematode control. The purpose of this study was to identify chemical compounds in crude extract of Macrotyloma axillare 'Java' and investigate their inhibitory effects on Meloidogyne javanica . Crude methanolic extract was fractionated into hexane, dichloromethane, ethyl acetate , and hydromethanol fractions. Chemical characterization of dichloromethane, ethyl acetate, and hydromethanol fractions was performed by dereplication using high-resolution mass spectrometry. The inhibitory activity of samples on the hatching of second-stage juveniles (J2) was tested at concentrations of 0%, 0.05%, 0.5%, and 5% (0, 500, 5000, 50000 ppm) and on nematode penetration and reproduction on soybean at 0.1% (1000 ppm). Distilled water was used as control in nematicidal assays. Several compounds belonging to the groups of organic acids, phenolic acids , fatty acids, flavonoids , pterocarpans, terpenoids, and sugars were identified. All fractions and concentrations reduced J2 hatching, reaching a plateau at 0.05%. Crude methanolic extract and dichloromethane and ethyl acetate fractions decreased M. javanica penetration at 10 days after inoculation, and all fractions reduced nematode penetration and female numbers at 30 days after inoculation. Extract fractions also reduced total nematode numbers, providing 49–95% control of the nematode. M. axillare 'Java' extract is rich in nematicidal compounds and has toxic effects on M. javanica , inhibiting J2 hatching and nematode penetration, development, and reproduction on soybean. • Macrotyloma axillare 'Java' is a plant that can be used to control nematodes. • 'Java' extract reduces penetration, development and reproduction of Meloidogyne javanica in soybean. • 135 compounds were identified in the 'Java' extract. • The chemical compounds of 'Java' show toxicity to Meloidogyne javanica .