This study was conducted to assess in-vivo antibacterial action of different levels of zinc oxide nanoparticles (ZnO NPs) against E. coli-induced infection in broilers. At 12 days old, 90 birds were randomly divided into six groups: two controls and four treatment groups. On day 19, all groups except the negative control were infected with E. coli. Once signs of colibacillosis appeared, birds received oral ZnO-NPs at varying doses and colistin. After six days of treatment, the birds were slaughtered for sample collection on the 8th and 11th days post induced infection. Mortality rate, hematological, biochemical, and immunological parameters, as well as gross and histopathological changes in immune organs, were assessed. Statistical analysis was conducted to compare differences among the groups. There was non-significant (P>0.05) difference of NDV titers among all the groups. Hematology, biochemical parameters, lymphoproliferative response and phagocytic index were significantly (P<0.05) higher in groups treated with zinc oxide NPs 100 mg/kg than control positive group. The mortality rate, gross pathology and quantitative histopathology indicated the better response in the 100 mg/kg ZnO-NPs treated group. E. coli induced infection in broilers can be treated with zinc oxide nanoparticles @ 100 mg/kg.