The current study investigated the effects of zinc oxide nanoparticles (ZONPs) and oxytetracycline (OTC) supplementation on broilers’ behavior, performance, carcass quality, biochemical parameters, and intestinal microbial populations and birds’ response to Newcastle disease (ND) vaccine. A total of 336 seven-day-old IR broiler chicks were randomly allotted to six dietary treatments containing 0, 10, 20, 30 and 40 ppm ZONPs or 50 ppm OTC. Each diet was fed to 7 replicates (8 birds/pen). The results clarified that 10 ppm ZONPs significantly improved the body weight gain and feed conversion in comparison to the control. No changes in behavior were recorded. The 10 ppm and 30 ppm ZONPs and OTC significantly reduced the gizzard weight in comparison to the control. While, 10 ppm ZONPs significantly increased the spleen weight, and all ZONPs doses increased bursa weight in comparison to the control and OTC groups. 20 ppm ZONPs increased the eviscerated yield and edible yield in comparison to the control and OTC groups. 40 ppm ZONPs increased pH, reduced meat color and overall acceptability in comparison to the control. In addition, results revealed that the 20 ppm ZONPs increased Calcium (Ca), High density low cholesterol (HDL-C), reduced urea (UA) and triglyceride (TG). Also, 40 ppm ZONPs and OTC increased creatinine (Cr) and reduced ND-HI titer in comparison to the control. For microbial population, OTC group was significantly lower than ZONPs groups in the total anaerobic, aerobic and lactobacilli count. In conclusion, the dietary inclusion of ZONPs can be applied as antibiotic growth promoter substitutions in broilers’ diet. However, further investigations are still needed.