BACKGROUND: Sorghum anthracnose, caused by Colletotrichum sublineola, poses a major threat to sorghum production, causing substantial damage globally. Linalool, a terpenoid synthesized by plants, plays a crucial role in defense against pathogens. This study explored the dual defense roles of the plant-synthesized terpenoid linalool through both host resistance potentiation and direct antifungal action.RESULTS: Following fungal infection, linalool levels increased significantly in sorghum leaves, with resistant genotypes exhibiting higher linalool accumulation than susceptible ones. A strong positive correlation between linalool content and disease resistance suggested its importance in fungal defense. Transcriptomic profiling identified Sobic.004G019400 as the key terpene synthase (TPS) gene responsive to fungal infection. This was validated functionally through heterologous expression and gas chromatography-mass spectrometry (GC-MS) analysis. Physiological assessments demonstrated that linalool application reduced lesion areas and alleviated declines in photosynthetic activity and transpiration in infected plants. The study also indicated that linalool inhibited C. sublineola spore germination and hyphal growth. Mechanistic investigations demonstrated that linalool effectively inhibited hyphal growth by inducing malondialdehyde overaccumulation, which caused significant damage to the fungal cell membrane and structure. This damage was evidenced by increased electrical conductivity and nucleic acid leakage. Transcriptomic analyses further revealed that linalool disrupted pathways in C. sublineola, including oxidative phosphorylation and the tricarboxylic acid (TCA) cycle.CONCLUSION: These findings not only identify Sobic.004G019400 as a molecular target for breeding anthracnose-resistant sorghum varieties, but also establish linalool as a functional antimicrobial agent with potential applications for integrated pest management strategies. © 2025 Society of Chemical Industry.