This study aimed to evaluate the effect of extrusion and of open-pan cooking on whole germinated and non-germinated grains of pearl millet (Pennisetum glaucum L. R. Br.), on its chemical-nutritional composition and in vitro iron bioavailability. The experimental design consisted of three flours: non-germination open-pan cooked millet flour (NGOPCMF), germination open-pan cooked millet flour (GOPCMF), and extrusion cooked millet flour (ECMF). The ECMF increased the carbohydrates, iron, manganese, diosmin, and cyanidin and decreased the total dietary fiber, resistant starch, lipids, and total vitamin E, in relation to NGOPCMF. The GOPCMF increased the lysine and vitamin C and decreased the phytate, lipids, total phenolic, total vitamin E, and riboflavin concentration, in relation to NGOPCMF. Furthermore, germinated cooked millet flour and extruded millet flour improved iron availability in vitro compared to non-germinated cooked millet flour. GOPCMF and ECMF generally preserved the chemical-nutritional composition of pearl millet and improved in vitro iron bioavailability; therefore, they are nutritionally equivalent and can be used to develop pearl millet-based products.