Ten eudesmanolides (1-10) including five new ones (1-4 and 6) were isolated from the whole plant of Wedelia trilobata (L.) Hitchc., a notoriously invasive weed in South China. As main constituents of W. trilobata, eight of these eudesmanolides were tested for their antitobacco mosaic virus (TMV) activities by the conventional half-leaf and leaf-disk method along with Western blot analysis. All of the tested compounds, at 10 μg/mL, showed strong antiviral activities in the pretreated tobacco plants with inhibition rates ranging from 46.7% to 76.5%, significantly higher than that of the positive control, ningnanmycin (13.5%). Their potential of inducing systemic acquired resistance (SAR) was also evaluated, and compounds 1 and 8 showed excellent induction activities. Furthermore, it was found that different concentrations of compound 1 promoted phenylalanine ammonia-lyase (PAL) activity in tobacco plants. To our knowledge, this is the first report that eudesmanolides could induce resistance in tobacco plants against the viral pathogen TMV.