Lignocellulosic biomass is a very attractive substrate for biogas production via anaerobic digestion. Among all, straw represents a very interesting and worldwide diffused agricultural by-product. However, due to the intrinsically complex structure of lignocellulosic biomass, straw has low biodegradability which results in low biogas yield. To increase the biogas production, chemical and physical pretreatments have been performed – i.e., size reduction, autoclave, and oxidation. The pretreatment conditions have been mitigated, in order to reduce their economic impact on the overall process and to make such pretreatments attractive at industrial level. The effects of the biomass pretreatments have been evaluated both by assessing the biomethane productivity in an anaerobic bioreactor and, in parallel, by characterizing the biomass at different levels – elemental content, functional groups, structural changes, and surface morphology. Results show a poor correlation between biogas production and the structural and chemical biomass changes. These findings confirm a more general issue: the difficulty of using biomass characterization alone to explain and predict the biogas production enhancement and of using such information to further improve biomass pretreatments.