The isoquinoline alkaloids have attracted widespread attention from chemists and biologists due to their complex structures and prominent bioactivities. With the aims of screening for antiviral compounds produced by fungi, in this study, three new (1-3), together with four known (4-7) isoquinolines were isolated from the cigar tobaccoderived endophytic fungus Aspergillus felis. Their structures were determined by means of HRESIMS and NMR spectroscopic studies. Interestingly, an anti-TMV test revealed that compounds 2 and 4 showed high activity with inhibition rates of 43.2% and 38.4%, respectively. These rates are higher than those of the positive control ningnanmycin (33.2%). The isolation of the above isoquinolines may provide materials for the study of more potent anti-TMV inhibitors and contribute to an improved utilization of cigar tobacco-derived endophytic fungi.