Biochemical interactions between biocontrol agents and the production of bioactive metabolites play a crucial role in interspecies signaling and the suppression of plant pathogens. In this study, the bioactive potential of metabolites extracted from monocultures and co-cultures of the nematophagous fungus Arthrobotrys oligospora and the bacterium Bacillus subtilis was evaluated. Metabolomic analyses conducted using LC-MS and GC-MS techniques demonstrated that microbial co-cultivation generated a broader metabolic diversity compared to monocultures. The presence of compounds such as cyclic dipeptides and bacterial antibiotics was a key determinant in shaping these interactions. Evaluation of pathogenicity and growth indices revealed that the NBA treatment significantly reduced the number of galls from 129 to 56 and egg masses from 101 to 38, while increasing root dry weight from 0.14 g to 0.50 g and significantly improving shoot growth. Furthermore, this treatment preserved plant protein and carbohydrate contents compared to the nematode-alone N treatment through the modulation of phenylalanine ammonia-lyase activity and the prevention of stress-induced accumulation of hydrogen peroxide and total phenols. Overall, the findings of this research confirm that interspecies interactions under co-culture conditions induce the biosynthesis of novel bioactive compounds, highlighting the high potential of microbial co-cultivation as an efficient strategy for discovering emerging bioactive molecules and developing sustainable approaches for the management of plant-parasitic nematodes.