Sorghum biscuits were enriched with mushroom powders (Lentinula edodes, Auricularia auricula and Tremella fuciformis) at 5%, 10% and 15% substitution levels. An in vitro gastrointestinal digestion was used to evaluate the effect of this enrichment on the phenolic content and soluble peptide content as well as antioxidant activities of the gastric or intestinal supernatants (bio-accessible fractions), and the remaining portions of phenolic compounds, antioxidants and β-glucan in the undigested residue (non-digestible fraction). The phenolic content of the gastric and intestinal supernatants obtained from digested mushroom-enriched biscuits was found to be higher than that of control biscuit, and the phenolic content was positively correlated to the antioxidant activities in each fraction (p < 0.001). L. edodes and T. fuciformis enrichment increased the soluble protein content (small peptide) of sorghum biscuits after in vitro digestion. All mushroom enrichment increased the total phenolic content and β-glucan content of the undigested residue and they were positively correlated (p < 0.001). The insoluble dietary fibre of biscuits was positively correlated with β-glucan content (p < 0.001) of undigested residue. These findings suggested that enriching food with mushroom derived dietary fibre increases the bioavailability of the non-digestible β-glucan and phenolic compounds.