With the aim of screening more antiviral activity metabolites, in this study, two new (1 and 2), together with five known (3-7), isoquinolines were isolated from the cigar tobacco-derived endophytic fungi Aspergillus fumigatus. Their structures were determined by means of HR-ESI-MS and extensive 1D and 2D NMR spectroscopic studies. Interestingly, the anti-TMV activities test revealed that compounds 1 and 2 showed potential anti-TMV activities with inhibition rates of 31.2 and 28.5%, respectively.