Oyster mushrooms (Pleurotus spp.) produced powerful bioactive peptides that slowed carbohydrate breakdown by blocking two key enzymes, α-amylase and α-glucosidase. Enzymatic processing boosted this inhibitory activity by up to 72%, and the peptides remained active even after simulated digestion. Both large and small peptide fractions were effective, positioning oyster mushroom proteins as a natural tool for moderating post-meal blood sugar spikes.