Two new sesquiterpenes, nicotianasesterpenes A and B (1 and 2), together with five known sesquiterpenes (3–7) were isolated from the leaves of Nicotiana tabacum. Their structures were determined mainly by spectroscopic methods, including extensive 1D- and 2D-NMR techniques. The anti-tobacco mosaic virus (anti-TMV) activities of compounds 1–7 were evaluated. The results revealed that compound 1 exhibited high anti-TMV activities with inhibition rates of 33.6%. This rate is high than that of positive control. The other compounds also showed potential activities with inhibition rates in the range of 18.8–28.4%, respectively.