Abstract: To investigate the roles of 6-methoxy-benzoxazolin-2-one (MBOA) and benzoxazolin-2-one (BOA) in the health-promoting effects of whole-grain foods, this study evaluated their in vitro antioxidant capacity and modulatory effects on gut microbiota. The results demonstrated that while MBOA and BOA exhibited dose-dependent antioxidant activity, their efficacy was inferior to that of ferulic acid. Compared to equivalent doses of ferulic acid, MBOA, BOA, and the mixture significantly enhanced microbial diversity in a dose-dependent manner, with the mixture showing optimal efficacy. At the phylum level, these compounds significantly reduced the relative abundances of Proteobacteria and Bacteroidetes while increasing those of Firmicutes and Actinobacteria. At the genus level, they markedly elevated the relative abundances of Bifidobacterium and reduced Escherichia, Prevotella, and Bacteroides. This study indicates that MBOA and BOA in whole grains possess substantial prebiotic potential.