Maintaining grape cultivation requires the continuous production of healthy propagating material and the control of economically important viruses. For more effective virus eradication, it is beneficial to try chemotherapeutic agents that have not yet been used on grapevine. We therefore tested two chemotherapeutic agents with different mechanisms of action (2-thiouracil and zidovudine) in addition to ribavirin, which is already used on grapevine. Viruses and viroids were identified in the mother plants of different grapevine cultivars by small RNA HTS (High-Throughput Sequencing), and RT-PCR verified the results. After chemotherapy, the regenerated plants were tested using RT-PCR and the efficacy of the three chemotherapeutic compounds was evaluated. Among the tested agents, ribavirin had the broadest elimination effect (the virus was not detectable by RT-PCR after 8 months) on most viruses. It proved to be particularly effective (the virus was not detectable by RT-PCR in most of the tested plants) against GFkV, GLRaV-4, GPGV and GRSPaV. The use of 2-thiouracil caused high phytotoxicity and was effective against GLRaV-4 among the viruses tested, having no effect on the other viruses. Zidovudine alone failed to eliminate any of the viruses we tested. 2-thiouracil, ribavirin and zidovudine were unable to eliminate HSVd and GYSVd-1 viroids.