The Paleo-Tokyo Bay (Kanto Plateau) that persisted during the transgression periods in the Middle-Late Pleistocene is known for its rich marine fauna that accommodated both the warm and cold water taxa brought by the Kuroshio and Oyashio Currents, respectively. However, little is known on the paleoichthyofauna in this area and the processes shaping the marine ichthyofauna of modern southern Kanto. We used fossil fish otoliths and teeth discovered in the Sha’ana Tuffaceous Sand Member belonging to the Chibanian Miyata Formation, exposed at Sha’ana-dai, Miura City, Kanagawa Prefecture, Japan, to reconstruct the paleoichthyofauna and its associated paleoenvironment and explore the content of fish assemblages during this period. The Sha’ana Tuffaceous Sand Member was composed of upper sandy mud and lower muddy sand horizons at the exposure. A total of 1,675 fish remains were collected from this site, of which 1,389 were assigned to 62 distinct taxa (20 orders, 31 families). The taxonomic composition revealed taxa from various environments, including shallow continental shelf, epi- to meso-pelagic settings, and the majority were from a generally temperate climatic zone. Paleoenvironmental analyses suggested that the fish assemblages were from the continental shelf (approximate depth of 100–200 m) in a temperate sea where both warm Kuroshio and cold Oyashio Currents had an influence on the biota. The analysis also showed that two boreal fishes, namely Clupea pallasii and Theragra chalcogramma, once had a wider distribution that extended to the southwestern areas of the Paleo-Tokyo Bay. This study provides a more complete view of the related paleobiogeography of marine fishes during the Chibanian and is essential for reconstructing the evolutionary processes of the rich ichthyofauna in the southern Kanto.