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Upcycling brewers’ spent grain (BSG) into poultry feed needs to be optimized. Since broiler chickens inefficiently digest fiber, we created a water-soluble BSG extract (BSGE) to explore this fraction’s potential nutritional benefits. We utilized intra-amniotic administration (in ovo) to target the gastrointestinal tract of broiler embryos. BSGE increased villus surface area and goblet cell quantity and size, implying improved duodenal development. The extract also changed cecal Escherichia coli (E. coli) and Clostridium abundances. Synchrotron X-ray fluorescence microscopy, along with zinc and iron transporter relative expression, did not reveal significant changes by BSGE. These findings highlight the potential for BSGE to be a functional feed component, underscoring the potential value of upcycling this byproduct. This pilot study supports future work exploring the impact of BSGE within feed and its effects over long-term consumption.