This study aimed at evaluating the ability of 6-pentyl-α-pyrone (6PP) isolated from Trichoderma koningii CTX1172 (AUMC 11520), an endophytic fungal strain to induce systemic resistance in tobacco (Nicotiana tabacum cv. White Burley) plant against tobacco mosaic virus (TMV). 6PP exhibited 10–60% symptoms inhibition at low concentrations (10–30 μg mL−1) achieving 100% biocontrol efficacy at high concentrations (40 and 50 μg mL−1), compared with control. Using DAS-ELISA technique for detection of TMV (expressed by decreasing in absorbance value), the TMV detection was significantly reduced in plants treated with 6PP at low concentrations compared with plants inoculated with TMV only and it was almost undetectable in plants treated with high concentrations, as well as healthy control plants. Application of 6PP not only increased the accumulation of proline as a non-enzymatic antioxidant compound, but also increased the activities of pathogenesis-related enzymes (superoxide dismutase, peroxidase and polyphenol oxidase), indicating that 6PP acts as elicitor for induction of resistance in tobacco against TMV. On the molecular level, plants treated with 6PP also showed augmented and rapid expression of defense-related genes including PR-a, PR-b and PR-10. This study was the first to imply the potential of a pyrone compound in biocontrol of plant viral disease.