Escherichia coli (E. coli) infection has significant public health impact on both chickens and human. Antibiotic resist- ance as well as antibiotic residues in chicken meat are some of the negative outcomes of the traditional antibiotic-based approach to prevent and control bacterial infections. Therefore, the main goal of current investigation was to control the drug-resistant E. coli O6 infection using nano-production of zinc oxide (ZnO-NPs) in both in vitro and vivo studies. ZnO-NPs was applied in one day old specific pathogen free chicks to evaluate the antibacterial effectiveness of 50mg/kg ration dosage compared with colistin as com- mercial antibiotic at 5 days old. E. coli serotype O6 was the highest prevalent and pathogenic multi drug resistant bacterial strain.The assessment parameters were clinical signs, post-mortem lesions and histopathological picture which showed effective role of ZnO- NPs as bacterial inhibitor in the treated groups compared to control one. Quantitative analysis showed that ZnO-NPs significantly lowered gross lesion scores in the liver, cecum, colon, spleen, heart, and lungs compared to the E. coli-infected group. These findings solidified our central hypothesis which was to evaluate the antimicrobial and antioxidant efficacy of ZnO-NPs against pathogenic bacterial strain of E. coli in broiler chicken as a powerful, safe alternative to antibiotics.