Background: Ya-Ha-Rak (YHR) or Bencha-Loga-Wichian is a traditional Thai medicine of polyherbal formulation, comprising five roots, commonly used as an antipyretic and antiseptic. Dengue virus infection exhibits high fever, which is the most common sign of febrile diseases, not just dengue infection. However, thus far, no study has reported the efficacy of YHR against this virus. Therefore, this study aimed to screen in vitro the efficacy of YHR in killing dengue virus type 2 (DENV-2). Method: Water (aqueous-YHR) and ethanol (EtOH-YHR) were used for studying phenolic and flavonoid contents, antioxidant scavenging activity (DDPH and ABTS), and cytotoxicity. In addition, the in vitro DENV-2 killing efficacy of YHR extracts was examined by the quantity of the DENV-2 focus-forming units and copies of RNA in cell culture supernatants. Results: The total phenolic content and the antioxidant scavenging activity (DDPH and ABTS) of EtOH-YHR were significantly higher than the aqueous-YHR. However, the aqueous-YHR was lower in cellular cytotoxicity than the EtOH-YHR. Quercetin showed the highest concentration among the 10 polyphenolic contents in the aqueous-YHR and EtOH-YHR extracts. In addition, the concentration of quercetin in EtOH-YHR was found to be almost 2.5-fold higher than that of aqueous-YHR. The EtOH-YHR was found to have higher inhibitory efficacy for DENV-2 infection than aqueous-YHR. Conclusion: The results obtained show that EtOH-YHR is a potential traditional Thai medicinal plant used as an alternative therapeutic agent against the deadly Dengue virus. © 2022 Elsevier GmbH