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This study investigates the effectiveness of two types of prebiotics—stachyose and raffinose—which are present in staple food crops that are widely consumed in regions where dietary Fe deficiency is a health concern. The hypothesis is that these prebiotics will improve Fe status, intestinal functionality, and increase health-promoting bacterial populations in vivo (Gallus gallus). By using the intra-amniotic administration procedure, prebiotic treatment solutions were injected in ovo (day 17 of embryonic incubation) with varying concentrations of a 1.0 mL pure raffinose or stachyose in 18 MΩ H2O. Four treatment groups (50, 100 mg·mL−1 raffinose or stachyose) and two controls (18 MΩ H2O and non-injected) were utilized. At hatch the cecum, small intestine, liver, and blood were collected for assessment of the relative abundance of the gut microflora, relative expression of Fe-related genes and brush border membrane functional genes, hepatic ferritin levels, and hemoglobin levels, respectively. The prebiotic treatments increased the relative expression of brush border membrane functionality proteins (p < 0.05), decreased the relative expression of Fe-related proteins (p < 0.05), and increased villus surface area. Raffinose and stachyose increased the relative abundance of probiotics (p < 0.05), and decreased that of pathogenic bacteria. Raffinose and stachyose beneficially affected the gut microflora, Fe bioavailability, and brush border membrane functionality. Our investigations have led to a greater understanding of these prebiotics’ effects on intestinal health and mineral metabolism.