Fish kill incidents are not rare in the upstream region of the Han River where overall water quality is suitable for supporting a fish community. However, the causes of fish kills are often not identified. In this study we examined the cause of a fish kill in an upstream tributary of the North Han River (Seo Stream, Yanggu County). In the field survey at the time when the fish kill occurred, the results did not indicate any harmful concentrations of heavy metals or toxic pesticides. Instead several tons of gingko (Ginkgo biloba) seed coat wastes were found in the water along the reach of fish kill site. Because gingko is known to be toxic, it was suspected as a cause of the fish kill. Recently gingko production has been increasing in Korea, and it is common practice for farmers to wash the seed coats of seeds in streams because the seed coats are toxic and emit an offensive odor. Toxicity of gingko fruit to humans is well known, but the toxicity to aquatic biota has not been previously reported. We investigated the toxicity of gingko seed coat extract to an invertebrate (Daphnia magna) and a fish (Oryzias latipes). After 48 hours exposure to seed coat extract the EC50 of Daphnia immobilization was 0.35% of a mixture corresponding to a dissolved organic carbon (DOC) concentration of 413 mgC L−1. In the fish experiment, the LC50 after 96 hours exposure was 0.4% of the extract mixture corresponding to a DOC of 440 mgC L−1. Thus gingko seed coat is toxic to aquatic animals and gingko washing was the likely cause of the fish kill incident. Our results suggest that gingko seed coat waste should be treated properly and not disposed of in streams.