The ability of edible mushrooms to modulate the intestinal microbiota is a topic of interest. This study shows that digested Pleurotus djamor powder (MUS) exhibits prebiotic effects during an in vitro colonic fermentation. Phenolic compounds, including epicatechin (3.03 ± 1.54 mg/L), gallic acid (2.71 ± 1.54 mg/L), and quercetin 3-glucoside (2.40 ± 1.54 mg/L), were found in P. djamor. MUS significantly increased the relative abundance of Lactobacillus spp./Enterococcus spp. (1.12% − 4.83%), Bifidobacterium spp. (0.59% − 1.85%), Ruminococcus albus/R. flavefaciens (0.37% − 1.88%), and reduced Clostridium histolyticum (2.89% − 1.22%) during 48 of colonic fermentation. MUS enhanced lactic acid and short-chain fatty acid production and decreased pH levels. The 1 H NMR analysis revealed the presence of essential amino acids, branched-chain amino acids, and other compounds that benefit human health. The results indicate the prebiotic effects of P. djamor on human intestinal microbiota.