The purpose of this work was to optimize the pretreatment process of wheat straw by Polyporus brumalis_BRFM985 in order to improve carbohydrate accessibility for more efficient bioconversion. Indeed, there is growing demands to develop sustainable routes for lignocellulosic feedstocks valorization into value-added products in energy, chemicals, materials, and animal feed fields. To be achieved, implementation of cheap and ecofriendly biomass pretreatment processes is necessary. In this frame, white rot basidiomycetes, well known for their ability to degrade lignin efficiently and selectively, are of great interest. The pretreatment of wheat straw by Polyporus brumalis_BRFM985 was performed in packed bed bioreactor and optimized using response surface methodology. The four pretreatment parameters optimized were metals addition (Cu, Mn, and Fe), time of culture, initial water content, and temperature. Multicriteria optimization highlighted that wheat straw pretreatment by Polyporus brumalis_BRFM985 in the presence of metals with high initial water content of 3.6 g H2O/g at 27°C for 15–16 days led to an improvement of carbohydrate accessibility with minimal matter loss.