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Several phytopathogens attack eggplant, causing crop damage. One of the most destructive plant diseases, Root-Knot Nematode (RKN), causes significant damage to eggplant seedlings. Finding safe and effective biological alternatives to prevent eggplant root nematode disease, which significantly limits plant productivity, is the innovative aspect of this study. Six isolates of plant growth-promoting fungus (PGPF) were tested in the current work for improving biochemical defense and physio-biochemical performance in eggplant seedlings under the Meloidogyne incognita challenge. PGPF isolates were tested in vitro for some biochemical traits such as Siderophores and HCN production. Besides, the antagonistic efficacy of PGPF filtrates against M. incognita was tested in vitro. The best isolates capable of producing HCN were F5 and F3 respectively. Also, F5 followed by F3 exhibited the maximum mortality proportions of 74.20% and 60.35% mortality in nematode juveniles after 96 hours respectively. Moreover, F5 has the highest level of antioxidant activity, with IC50 145 µg/mL followed by F3 with IC50 350 µg/mL. thus, we identified F5 and F3 completely as Rhizopus microsporus (OQ291571.1 and Aspergillus oryzae OQ291572.1. Implementing R. microsporus and A. oryzae collectively in vivo study was the most successful therapy, limiting nematode recordings as 95.23%, 86.98%, 80.35%, 80%, and 68.78% reduction in females, galls, developmental stage, egg masses, second juveniles, respectively, in diseased seedlings. It could be suggested that the use of ethyl acetate extracts (EAE) of A. oryzae and R. microsporus might be commercially applied as a stimulator of eggplant and or anti-nematodes against M. incognita. [ABSTRACT FROM AUTHOR]