Daidzein is an isoflavone found primarily in soybean and various soy-based products such as tofu. In the intestines, daidzein is reductively transformed to its constituent metabolites equol and O-desmethylangolensin. Although the ingestion of daidzein has been associated with marked physiological improvements in various pathological states, namely cancer, metabolic syndrome, and cardiovascular disease, further understanding into the tissue-level effects of daidzein ingestion is needed. In this study, broiler chickens (Gallus gallus, n = 50) were injected in ovo (day 17 of embryonic incubation) with varying concentrations of a 1.0 mL pure daidzein in saline solution. Three treatment groups (0.5, 2.5, 5.0 mg mL⁻¹) and two controls (saline and non-injected) were utilized. Upon hatching, blood was taken for hemoglobin and total body iron hemoglobin determination, and chicks were then euthanized. Hepatic, duodenal, and caecal tissues were excised for ferritin analysis, mRNA gene expression of relevant brush border membrane and iron transporters/proteins, and PCR quantification of three bacterial genera (Bifidobacterium, Lactobacillus, and Clostridium) and one bacterial species (E. coli). Results revealed no significant differences in hemoglobin, total body hemoglobin iron, or ferritin between the groups (p > 0.05). Aminopeptidase and Na⁺K⁺ATPase were upregulated in daidzein-treated groups when compared to controls (p < 0.05). Additionally, daidzein administration increased the expression of Dcyt B, an iron-specific cytochrome reductase (p < 0.05). Further, daidzein injection resulted in the increased caecal abundance of E. coli in the 2.5 mg mL⁻¹ group (p < 0.05). Taken together, these results suggest a physiological role for daidzein administration in improving the functionality and development of the brush border membrane, as well as for influencing the abundance of certain bacterial communities in the colon.