Abstract: To clarify the effect of co-infection with alfalfa mosaic virus (AMV) and white clover mosaic virus (WCMV) on three phenolic substances (chlorogenic acid, total phenols and flavonoids) in Nicotiana rustica, N. rustica inoculated with AMV and WCMV separately and combined in a ratio of 3∶1 was set as the treatment, while N. rustica inoculated with PBS buffer alone served as the negative control. Symptoms of N. rustica were observed and DAS-ELISA was performed 6, 18 and 30 days after inoculation. At the same time, the leaves of N. rustica were collected 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 days after inoculation with viruses to determine the contents (mass fractions) of chlorogenic acid, total phenols and flavonoids. The results showed that the symptoms of N. rustica after co-infection with the two viruses were more severe than after single infection, showing plant dwarfism, leaf shrinkage and chlorosis. The average reproduction rate after co-inoculation with AMV and WCMV (30.68% and 28.24%) was lower than that after single inoculations (32.19% and 30.27%). After co-inoculation with AMV and WCMV, the contents of chlorogenic acid, total phenols and flavonoids in leaves of N. rustica infected were significantly higher than those inoculated with the single virus. The contents of the three substances reached maximum values of 558.98, 177.52 and 33.98 µg·g^-1 FW at 30, 27 and 30 d after inoculation, respectively. The maximum contents of chlorogenic acid, total phenolic and flavonoids in leaves of N. rustica after co-infection increased by 98.28%, 85.90% and 107.76%, 103.73%, 42.78% and 137.25%, 39.03%, 40.93% and 63.84% compared to single infection with AMV, WCMV and CK and the differences were significant. The results showed that the production of chlorogenic acid, total phenols and flavonoids in N. rustica after co-infection with the two viruses was correlated with the co-infection stress of AMV and WCMV.