With the aims of screening more antibacterial 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 puniceus. Their structures were determined by means of HR-ESI-MS and extensive 1D and 2D NMR spectroscopic studies. The new compounds were evaluated for antibacterial activities against Pseudomonas syringae (the main pathogenic sources of tobacco angular leaf spot disease), and compounds 1 and 2 exhibited inhibitory effects with MIC50 values of 8.5 and 5.4 μg/mL respectively. These rates are lower than that of positive control (agricultural streptomycin, with MIC50 of 2.2 μg/mL).