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Brewer’s spent grain (BSG), the main by-product of the brewing industry, is a rich source of arabinoxylans (AXs) and hydroxycinnamic acids, particularly ferulic acid (FA), which contribute to its prebiotic potential. This study evaluates the prebiotic properties of residual solid from an enzymatically treated BSG, compared to the properties of BSG as a non-enzymatically hydrolyzed control. Although the residual solid exhibited total polyphenol (2581.96 ± 70.63 mg/100 g dry weight) and FA (180.84 ± 3.28 mg/100 g dry weight) contents comparable to those of the non-hydrolyzed control (2500.38 ± 284.20 and 179.59 ± 3.30 mg/100 g dry weight, respectively), the AX content was significantly higher (14,084.81 ± 185.72 mg/100 g), accompanied by a lower degree of feruloylation (12.84 ± 0.23 mg FA/g AX), higher antioxidant activity (64,825.35 ± 4011.24 μmol TE/100 g), and structural changes visualized by scanning electron microscopy. In addition, in vitro colonic fermentation showed a delayed butyrogenic profile, with increased butyrate production compared to the control (3.17 ± 1.44 mM). Microbiota analysis by fluorescence in situ hybridization (FISH) coupled with flow cytometry indicated an increase in butyrate-producing bacteria, including Faecalibacterium prausnitzii (+5.90) and Eubacterium rectale (+6.88). Growth of Bacteroides, Lactobacillus, and Bifidobacterium spp. was also promoted. Overall, these findings suggest that enzymatic processing of BSG can generate a residual solid with modified structural characteristics and potential prebiotic functionality, supporting its potential application as a fermentable flour ingredient in functional foods.