This study compared the effects of hot-air pasteurisation (HAP) at 75–100 °C versus autoclaving at 121 °C and 2 bar overpressure on the lignocellulosic degradation process of birch-based substrates that were used for shiitake mushroom cultivation and potential bioethanol production. Fifty substrate samples were obtained as a time series from different stages of the cultivation, and their chemical contents were measured by chemical analysis and near infra-red spectroscopy (NIR). Despite of different energy intensities, HAP and autoclaving did not result in significant differences in the degradation of lignin and carbohydrates. Major compositional changes were associated with the cultivation process. Principal component analysis on the wet chemical data and orthogonal projections to latent structures based on NIR spectra reached the same conclusion, namely that HAP had similar effect as autoclaving on compositional changes in the substrate during cultivation. The results of this study suggest that a substitution of autoclaving by HAP may potentially save up to 9.9 TWh energy for the global production of 7.5 million ton shiitake. At the same time, lignocellulose feedstock can be pretreated for the production of up to 3.24 million m3 of 95%-ethanol fuels, which can potentially substitute proximate 1.88 million m3 of regular gasoline.