Novel chemical-independent approaches for suppression of plant parasitic nematodes are urgently needed. Anaerobic liquid digestates from biogas production have demonstrated suppressive capacity of plant-parasitic nematodes and may serve as bio-fertilizers. In this study, effects of substrates and fermentation conditions on digestate’s properties were investigated. In different experimental contexts, drench-applied digestates, derived either from food waste or dairy manure, processed mesophilically or thermophilically, were tested for suppressive potential of plant-parasitic nematodes. In radish bioassays, root penetration by Heterodera schachtii J2 was reduced when exposed to thermophilic digestates, and was 10-times less than the control in food waste digestate at 2 ml/35 ml soil. In a watermelon bioassay with Meloidogyne incognita, and a pot experiment with peach and Pratylenchus vulnus, thermophilic digestates improved plant growth more than mesophilic digestates. In the watermelon test, nematode suppression was stronger in thermophilic than mesophilic digestates, and higher in food waste rather than manure substrate. In a microplot trial with bell peppers and M. incognita, both digestates had some negative effects on plant growth. In summary, thermophilic fermentation and the food waste were the most effective process and substrate, respectively. Further adjustments are necessary to identify application rates that suppress nematodes without negatively effecting plants.