In the world, Ralstonia solanacearum, the second most harmful bacterial phytopathogen, poses a serious danger to potato crop by causing brown rot disease, which can result in losses of up to 100%. For that, this study aims to isolate and identify of pathogenic bacteria causing brown rot disease and biocontrol it using ZnONPs synthesized by fungal strain. Eighteen infected potato cv. Cara samples were collected from different six regions of El-Minya governorate, Egypt. Isolation, identification, and pathogenicity tests were done for the most common pathogenic bacteria. Also, 18 native agricultural soil samples from 6 regions of El-Minya governorate, Egypt. Additionally, isolation and identification of the most common fungal isolate can synthesize ZnONPs. Mycosynthesized ZnONPs were characterized by UV, TEM, SEM, EDX, XRD, FTIR, Zeta potential, and particle size. Cytotoxicity test for mycosynthesized ZnONPs was done to determine the safety level of obtained nanoparticles which its dominant spherical or hexagonal shapes and small in size at 42.2 ± 3.0 nm. Results showed that the pathogenic bacterial isolate was identified using morphological, biochemical, and molecular as Ralstonia solanacearum (OR533702). Also, 16 fungal isolates were isolated and the most common was selected and identified as Epicoccum nigrum (OR533699) using microscopic, macroscopic, and molecular identification. Additionally, the result showed the selected fungal strain E. nigrum has high efficacy with inhibition zone diameter (2.20 ± 0.20) mm at 100 μg/ mL (5mM) against R. solanacearum. This study concluded that mycosynthesized ZnONPs have a potent effect against R.solanacearum compared to Revanol 50% as a traditional product.