Two new β-carboline alkaloids, 1-acetyl-6-hydroxymethyl-7-methoxy-β-carboline (1) and 1-acetyl-6- hydroxymethyl-8-methoxy-β-carboline (2), together with four known ones (3–6) were isolated from the cigar tobacco (a variety of Nicotiana tabacum L.)-derived endophytic fungi Aspergillus sp. Their structures were determined by means of HR-ESI-MS and extensive 1D and 2D NMR spectroscopic studies. The anti-TMV activities test revealed that compounds 1 and 2 showed potential anti-TMV activities with inhibition rates of 31.8 and 35.5%, respectively. These rates are close to that of positive control. Compounds 3–6 also showed anti-TMV activities with inhibition rates in the range of 21.5–28.8%, respectively. The isolation of the aforementioned β-carboline alkaloids may provide materials for the screening of anti-TMV activity inhibitors and contribute to the utilization of cigar tobacco-derived endophytic fungi. [ABSTRACT FROM AUTHOR]