This work aims to evaluate the antibacterial activity of biological zinc nanoparticles (BIO-ZnONPs) against pathogenic fish bacteria and assess the effect of BIOZnONPs 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 < 0.05). The liver function, LYZ activity, and NBT values were significantly enhanced in the 0.5 mg L-1 BIO-ZnONPS group compared to CH- ZnONPs group and control (P < 0.05). Furthermore, the lowest cortisol and the highest testosterone and growth hormone levels were recorded in 1mg L-1 BIO-ZnONPs group. Regarding the antibacterial effects, BIO-ZnONPs displayed the lower total bacterial loads in water and fish tissues (intestine, gills, skin, and muscle) and the maximum antibacterial properties against pathogenic bacteria (Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Aeromonas hydrophila). Our study 3 exemplifies novel findings of BIO-ZnONPs in the promotion of fish health and production and its antibacterial properties in Nile tilapia. Highlight: • Biological ZnONPs have considerable bactericidal against pathogenic fish bacteria. • Biological ZnONPs reduced the bacterial load in water and fish tissues. • Biological ZnONPs enhanced FCR feeding and swimming behaviors. • Biological ZnONPs stimulated fish health and production. Keywords: ZnONPs; Biological; Physiological changes; Antimicrobial properties; Nile tilapia