This work aims to evaluate the antibacterial activity of biological zinc nanoparticles (BIO-ZnONPs) against pathogenic fish bacteria and assess the effect of BIO-ZnONPs on the performance, behavior, and immune response in Nile tilapia (Oreochromis niloticus) as compared to chemical zinc nanoparticles (CH- ZnONPs). Aspergillus niger TS16 fabricated the BIO-ZnONPs were spherical shape with the average size of 45 nm and net charge of −27.23 mV. Generally, the results indicate that BIO-ZnONPs were more effective than CH- ZnONPs in enhancing the performance properties of Nile tilapia. Five experimental groups of Nile tilapia (initial body weight of 20.2 g) were treated with two concentrations of 0.5 and 1 mg L−1 from biological and chemical ZnONPs, while the fifth group was served as a control. After ten weeks of treated water with ZnONPs, the performance, feed efficiency parameters, feeding, and swimming behaviors significantly improved in BIO-ZnONPs treated groups (P