The chromone alkaloids have attracted the widespread attention of chemists and biologists due to their complex structures and prominent bioactivities. With the aim of screening more antiviral activity metabolites, in this study, two new (1 and 2), together with five known (3–7) chromone alkaloids were isolated from the cigar-tobacco-leaf-derived endophytic fungi Aspergillus lentulus. Interestingly, the anti-TMV activities test revealed that compounds 2, 5, and 7 showed high anti-TMV activities with inhibition rates of 44.2%, 38.5%, and 40.2%, respectively. These rates are higher than that of the positive control. Compounds 1, 3, and 5 also showed potential anti-TMV activities with inhibition rates in the range of 28.4–31.8%, respectively. The isolation of the aforementioned chromone alkaloids may provide materials for the screening of anti-TMV activity inhibitors and contribute to the utilization of the cigar-tobacco-derived endophytic fungi. © 2023, Springer Science+Business Media, LLC, part of Springer Nature.