Brewer's spent grain (BSG) is a waste product of the beer industry, and γ-aminobutyric acid (GABA) is a physiologically active substance important for brain and neuron physiology. In this study, we used the bacterial strains Bacillus velezensis DMB06 and B. licheniformis 0DA23-1, respectively, to ferment BSG and produce GABA. The GABA biosynthesis pathways were identified through genomic analysis of the genomes of both strains. We then inoculated the strains into BSG to determine changes in pH, acidity, reducing sugar content, amino-type nitrogen content, and GABA production, which was approximately doubled in BSG inoculated with Bacillus compared to that in uninoculated BSG; however, no significant difference was observed in GABA production between the two bacterial strains. These results provide the experimental basis for expanding the use of BSG by demonstrating the potential gain in increasing GABA production from a waste resource.