The influence of in ovo administration of grape seed extract (GSE) and vitamin C on hatchability, glutathione peroxidase activity (GPx), yolk sac weight, performance and ileal microflora of broiler chickens were investigated in broiler. A total of 490 fertile eggs were divided into seven groups: control group (not punctured nor treated with additive), control group (penetrated with no additive), sham group (normal saline, 0.5 ml/egg), GSE (3 mg/egg), GSE (4.5 mg/egg), GSE (6 mg/egg) and vitamin C (3 mg/egg). Experimental preparations were injected into eggs at day 18 of incubation period. Hatched chicks were raised till 10 days of age. In ovo injection of 4.5 mg GSE/egg significantly increased hatchability and GPx activity. However, there were no significant differences among day-old weight, yolk sac weight and mortality of chicks. In ovo administration of 4.5 mg GSE or vitamin C increased average daily weight gain and average daily feed intake of chickens compared to control groups. Further, In ovo injection of 4.5, 6 mg GSE or 3 mg vitamin C decreased ileal population of Coliforms and E. Coli. In conclusion, in ovo injection of 4.5 mg GSE/egg injected on 18^th d of incubation has positive effect on broiler chickens and has no adverse effect on the performance of birds during starter period.