Wheat flour and durum semolina were enriched with powders from four medicinal mushrooms during dough mixing. Wheat flour and durum semolina were substituted with chaga, Cordyceps sinensis, reishi and lion’s mane powders at 3%, 6%, 9% and 12% (w/w). Changes in the gluten secondary structure and water populations of the dough samples were determined by means of Fourier transform infrared spectroscopy (FT-IR). As a result of mushroom powder incorporation, the formation of additional pseudo-β-sheets (aggregated structures) was observed in wheat flour doughs, whereas aggregates and additional α-helices were formed in the durum semolina doughs. Structural differences observed between dough materials can be due to the gluten index and particle size. Generally, chaga, reishi and lion’s mane powders caused an increase in the content of α-helices and a decrease in the content of pseudo-β-sheets, parallel-β-sheets and antiparallel-β-sheets. C. sinensis powder induced the formation of pseudo-β-sheets from α-helices, β-turns and antiparallel-β-sheets. The content of the mushroom powders in wheat flour and durum semolina doughs slightly affected the changes observed in the secondary structure of gluten. Amounts of particular water populations did not change or changed slightly in the presence of medicinal mushroom powders, which was presumably due to complex chemical composition of the mushroom powders. Analysis of the FT-IR spectra showed that the type of the observed structural changes depended on the material type the dough was prepared from as well as chemical composition of the mushroom powder.