The isoquinoline alkaloids have attracted widespread attention from chemists and biologists. With the aims of screening for biological compounds produced by fungi, three new (1-3), together with four known (4-7) isoquinolines were isolated from the cigar tobacco-derived endophytic fungus Aspergillus puniceus in this study. Their structures were determined by means of HRESIMS and NMR spectroscopic studies. Interestingly, the antifungal activity test revealed that compound 5 showed high antifungal activity against G. cichoracearum (DC) (the main pathogen of tobacco powdery mildew disease) with an inhibition rate of 76.3% +/- 5.0. Compared to the negative control, compounds 1-4, 6, and 7 also showed notable anti-G. cichoracearum activities with inhibition rates in the range of 38.5-56.9%. The isolation of the above isoquinolines may provide materials for the study of more potent antifungal inhibitors and contribute to an improved utilisation of cigar tobacco-derived endophytic fungi.[GRAPHICS].