Cultivation of edible mushrooms on straw can significantly reduce production costs, provide notable environmental and ecological benefits. However, the molecular mechanisms via which mushrooms utilize straw are not well understood. We conducted a comparative transcriptomic analysis of oyster mushrooms cultivated on two different biomass substrates, namely, corncob and tobacco straw at various developmental stages. The results revealed that the biomass substrates induced distinct transcriptomic changes during mycelium and fruiting body development. Straw affected the metabolism of pyruvate and acetyl-CoA, with significant downregulation of pyruvate decarboxylase expression, and upregulation of acetyl-CoA hydrolase during mycelial growth. Genes associated with cell wall and carbohydrate metabolism were significantly upregulated, and the PLD-PA-mTOR pathway was activated during the fruiting body development stage in mushrooms grown on straw. These findings reveal the transcriptomic adaptations of oyster mushrooms to tobacco straw, and enhance our understanding of the molecular mechanisms underlying biomass conversion by edible fungi using straw.