To explore the active metabolites from Aspergillus, we have discovered five undescribed tricyclic or tetracyclic indole alkaloids aspergillines K–O (1–5) and four known analogues (6–9) from the strain of Nicotiana tabacum-derived Aspergillus versicolorYNCA0363. The α-amino group derived from tryptophan in isolates 1–6was changed to an oxygen atom, which is uncommon in the reported analogues. To the best of our knowledge, 4and 5represent the first examples of indole alkaloids possessing an isochromene moiety. Compounds 1and 3showed obvious anti-tobacco mosaic virus (TMV) activities with inhibition rates of 43.6% ± 2.2 and 58.5% ± 2.8 at the concentration of 20 μM, respectively, which were significantly higher than that of the positive control. Compound 3also showed excellent potential of inducing systemic acquired resistance. Furthermore, compound 3can increase the activities of two reactive oxygen species-scavenging enzymes, peroxidase and superoxide dismutase, and enhance tobacco resistance to TMV. Molecular docking results showed that compound 3exhibits a stronger interaction with TMV-helicase, and showed higher antiviral activities than others. This work revealed that the above indole alkaloids, especially 3, could induce resistance in tobacco plants against the viral pathogen TMV.