Zinc oxide nanoparticles (ZnO-NPs) exhibit antimicrobial properties against most bacteria in many in vitro studies. However, in vivo validations of their antimicrobial effects in Nile tilapia have not yet been investigated. This study aimed to investigate the impacts of ZnO-NPs and their therapeutic effectiveness in experimentally infected Nile tilapia with S. parauberis. For safety assessment, 120 fish were divided into four groups. They were fed a diet supplemented with 0, 125, 250 and 500 mg/kg dry feed of ZnO-NPs for 7 days, respectively. Blood and tissue samples were collected to evaluate haematological, biochemical and histopathological alterations. Another 120 fish were divided into four groups. Group 1 (G1) served as the negative control, while G2, G3, and G4 were challenged intraperitoneally with S. parauberis. G2 was fed on a basal diet without medication. G3 and G4 were treated with ZnO-NPs (125 mg/kg basal diet) or ampicillin (50mg/kg basal diet) supplemented diet for 7 days. Fish were carefully observed for any abnormalities in fish behaviour, clinical alterations and mortalities for 16 days post-challenge. Blood and tissue samples were collected from different groups. Survival rates were 100% (G1), 46.67% (G2), 53.34% (G3), and 53.34% (G4). Several adverse changes in haematological, biochemical, immunological parameters and tissue histopathology were recorded in the challenged groups. Treatment of Nile tilapia with dietary doses of ZnO-NPs or ampicillin ameliorated the effect of S. parauberis infection. Despite the nontoxic impact of high dietary doses of ZnO-NPs on Nile tilapia, further improvement is needed to achieve a higher survival rate.