In recent decades, Zinc oxide nanoparticles (ZnONPs) are already used in several fields either for commercial or industrial purposes. In this study, 120 freshwater fish (Oreochromis niloticus) were divided into 4 different groups, each in triplicate groups (n=10), and used to assess the pharmacological activity of ZnONPs as an anti-bacterial and immunomodulatory agent.  Initially, the antibacterial activity was detected in-vitro through the disk diffusion method. The results revealed a potent antibacterial activity, especially at 80 mg/kg of ZnONPs. Then, Pathogenicity test with pseudomonas aeruginosa was done. The clinical signs following the infection were also detected and the mortality rate was calculated. Administration of ZnONPs (80mg/kg) showed potent ability to protect ‎the fish from mortality induced by I/P injection of P. aeruginosa. Finally,blood samples were collected twice, on the 20^th and 40^th days, to detect the effect of ZnONPs on some immune parameters including granulocyte, monocytes, lymphocytes and white blood cells (WBCs) count. The oral administration of both ZnONPs and zinc acetate resulted in a significant increase in lymphocyte, monocyte and WBCs count from the 20^th to 40^th days in ‎infected tilapia. In contrast, ‎ the results of granulocyte count revealed that the counts decreased from 20^th to 40^th days in groups exposed to 80mg/kg ZnO NPs. In conclusion, ZnO-NPs showed significant antibacterial activity against P. aeruginosa-infected tilapia fish either in-vitro or in-vivo as well as elevated the immunity of the fish against P. aeruginosa infection.