Three new isocoumarin derivatives, tabaisocoumarins A–C (1–3), together with three known phenolic compounds (4–6), were isolated from the roots and stems of Nicotiana tabacum. Their structures were elucidated by spectroscopic methods, including extensive 1D- and 2D NMR techniques. Compounds 1–6 were evaluated for their anti-tobacco mosaic virus (anti-TMV) activities at the concentration of 20 μM. Compound 2 exhibited anti-TMV activity with inhibition rate of 33.8%, which is higher than that of positive control, ningnanmycin. The other compounds also showed potential anti-TMV activities with inhibition rates in the range of 18.7–28.5%, respectively.