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Returning biogas digestate to the fields as biofertilizer is an established agricultural practice which has been promoting plant resistance to soil borne pests. Root-knot nematodes (Meloidogyne spp.) are widely distributed Plant Parasitic Nematodes (PPNs), constituting a major threat to the world’s food supply. Novel PPNs control methods are needed in the context of sustainable agriculture to replace or to complement the use of hazardous synthetic nematicides. The identification of nematicidal compounds in biogas digester effluents could provide a win–win solution for digestate’ costly disposal and for renewable crop-protection products formulation. However, the understanding of PPNs control mechanisms by digestate is still scarce. In this study, we evaluated the nematicidal activity of digestate samples from two on-farm biogas plants both in vitro and in planta on Meloidogyne incognita second stage juveniles (J2). Over 60 % J2 mortality was observed in vitro after only 24 h exposure to diluted digestate (10 % v/v), and reached > 90 % after 7 days. In greenhouse trials, digestate application at 10 % and 5 % dilution was effective in preventing root galling caused by M. incognita in highly susceptible cultivars (tomato and cucumber), sustaining plant biomass. Liquid–liquid fractionation(s) on the crude digestate mixture allowed the isolation of extracts of different polarity for untargeted compound analysis by Gas Chromatography–Mass Spectrometry (GC–MS). Our findings confirm the value of biogas digestate as PPNs suppressive amendment and introduce a novel approach to PPNs control, moving towards more circular and sustainable agri-food systems.