Betanodaviruses, members of the family Nodaviridae, are the causal agents of viral nervous necrosis (VNN) in many species of marine farmed fish. In the aquaculture industry, outbreaks of betanodavirus infection result in devastating damage and heavy economic losses. Although an urgent need exists to develop drugs against betanodavirus infection, there have been few reports about antibetanodavirus drugs. In this study, we examined the inhibitory effect of Oligonol, a purified phenolic extract from lychee fruit, on betanodavirus infection in fish cells. Oligonol significantly inhibited replication of betanodavirus (EC(50) = 0.9-1.8 μg/mL) as shown by the reduction of the virus-induced cytopathogenic effect (CPE) and the protection of cells in the crystal violet staining assay. The inhibition was dose dependent. A time-of-addition assay indicated that Oligonol's action takes place at an early stage of the viral infection. According to an attachment inhibition assay, it is possible that Oligonol partially inhibits attachment of the virion to the cell. Our data show that Oligonol could serve as an antiviral agent against betanodavirus.