This research was carried out to evaluated the effect of wheat flour substitution by different levels from whole barley flour (WBF), whole oat flour (WOF) and mixture (WBF+WOF) of them on chemical, rheological and organoleptic characteristics of Balady bread as well as its effect on lowering blood glucose level of hyperglycemic rats. The results showed that blending of WBF or/and WOF to wheat flour increased protein, fat, ash and crude fiber content in produced bread. Also, minerals concentration was increased gradually by increasing replacement levels of WBF and WOF in all tested samples compared with control. Rheological parameters such as water absorption, arrival time, dough development, dough stability and weakening were increased significantly (P ≤ 0.05). In contrast, elasticity, extensibility, proportion number and energy were decreased in all samples compared with control. Organoleptic properties of roundness and separation of layers for all tested bread samples were not affected significantly, while, a significant (P ≤ 0.05) difference was observed in taste, odor and crust color when WBF and WOF levels were increased over 20%. Results also revealed that the hyperglycemic rats fed on bread containing 20% WBF, WOF and (WBF+WOF) for 28 days caused a significant (P ≤ 0.05) decrease in blood glucose level compared with hyperglycemic rat's control. Thus, it can be suggested that combination of WBF or/and WOF in bread up to 20% gave good quality characteristics without adversely affecting the consumer acceptability of the bread and leads to lower blood glucose level in hyperglycemic rats.