Anchialine systems, or underground estuaries, are rare coastal ecosystems characterized by the mixing of groundwater and seawater, often hosting highly specialized and endemic species. In Okinawa, Japan, one of the world’s only two known “salt rivers” provides a unique opportunity to study interactions between hypogeal (underground) and epigeal (surface) components. This system features subterranean saltwater that mixes underground, is forced up through a spring, and flows 300 meters before emptying onto a fringing coral reef, creating distinct habitats. However, nearby open quarry mining activities might significantly threaten this ecosystem via sedimentation. We utilized eDNA metabarcoding and field surveys to characterize fish, invertebrates, and microbial communities within this Japanese natural monument. To characterize the site and to assess the impacts of sedimentation and water quality degradation from heavy rain events, we measured turbidity, salinity, temperature, and dissolved oxygen and performed chemical analyses spatiotemporally. Metabarcoding results revealed distinct fish species distributions along the surface and underground components. We further observed a clear correlation between rain events and a decline in water quality. Our findings highlight the need for effective management strategies to protect these rare ecosystems from ongoing anthropogenic pressures.