Tomatidine shows broad-spectrum antiviral activity against other swine disease viruses. TCID50, western blot, and qPCR were used to examine the inhibition activity of tomatidine against other swine disease viruses. Three designated concentrations of compounds were added to the culture medium (final concentrations were 2.5, 5, 10 μM, with no-observable cytotoxicity). DMSO was used as the negative control. TGEV (0.01 MOI), PRRSV (E), and EMCV (H)/SVA (K) were then used to infect ST, Marc-145, and BHK-21 cells, respectively, and samples were harvested at 24 h, 48 h, and 18 h. The TCID50 of TGEV (A), PRRSV (D), EMCV (G), and SVA (J) treated with 10 μM tomatidine or DMSO were calculated using the Reed-Muench method. The N-protein level of TGEV (B), PRRSV (E), EMCV (H), and SVA (K) were determined by western blot. Relative TGEV N (C), PRRSV N (F), EMCV VP1 (I), and SVA VP1 (L) mRNA levels, was determined by qRT-PCR, and expressed relative to that in DMSO-treated cells. M Viability of ST, Marc-145, and BHK-21 cells pretreated with the indicated concentrations of tomatidine and incubated for 24 h, 48 h, and 18 h, respectively in medium containing tomatidine. The results are from one of three independent experiments. The internal loading control was β-actin. Error bars represent the SD. The asterisks in the figures indicate significant differences (*P < 0.05; **P < 0.01; ***P < 0.001; ns = not significant).