The current study was performed to develop natural bio-active substances as additives for the production of high quality broiler chickens. A total of 120 male 3 day-old broiler chicks were randomly allocated to CON (control), GK2.5 (ginkgo leaf 2.5%), GK5.0 (ginkgo leaf 5.0%), PK2.5 (pumpkin 2.5%) and PK5.0 (pumpkin 5.0%) of five groups in cages (24 birds per group). All birds were fed corresponding diets from 3 to 35 d of age and determined growth performance and biological parameters including blood biochemical profiles, antioxidant status and intestinal microflora. During the entire feeding trial, GK5.0 and PK5.0 groups resulted in a significantly (P<0.05) higher FCR than GK2.5 and PK2.5 groups. Plasma triglyceride significantly (P<0.05) increased in GK5.0 group compared with the other groups, and the level of alanine transaminase (ALT) increased (P<0.05) in GK5.0 and PK5.0 groups compared with that in PK2.5 group. Dietary addition of ginkgo leaf and pumpkin significantly (P<0.05) increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the small intestine. Also, the addition of 2.5% ginkgo leaf significantly (P<0.05) increased the activities of SOD, GSH-Px and glutathione-S-transferase (GST) in the liver. Futhermore, muscle GST activity significantly (P<0.05) enhanced by dietary addition of ginko leaf and pumpkin. However, the level of lipid peroxidation (MDA) in the small intestine and muscle turned to be higher (P<0.05) in PK5.0 group. The colony forming units (CFU) of E. coli in intestinal digesta significantly (P<0.05) decreased in both ginko leaf and pumpkin supplemented groups compared with CON group. In conclusion, dietary addition of 2.5% ginko leaf and pumpkin as dietary sources can be applicable for the production of high quality broiler chickens.