Hospital Hole is a 43m deep stratified anchialine sinkhole with distinct freshwater and brackish water layers with anoxic conditions at depth. The sink is located in the Weeki Wachee River, FL. We measured the physicochemical parameters within each layer and characterized the corresponding microbial communities over the past 18 months. The surface river layer, which is <3 m deep, is characterized by high oxygen but low levels of salinity, sulfate, total organic carbon, sulfide, and ammonia. Stable isotope data (d18O) indicates this water is similar to other spring-fed rivers within the area. Below the river water is a brackish oxic layer between 3 m and 21 m depth, separated by a thermohalocline. This water is characterized by high levels of sulfate, lower levels of dissolved oxygen, and higher levels of salinity. At 21 m, a cloudy opaque chemocline is present. The chemocline varies in thickness from a few millimeters to 6 meters on different dates. This layer more closely resembles the oxic layer in terms of the geochemistry. Below the chemocline is an anoxic layer, characterized by complete darkness, anoxia, high concentrations of total organic carbon, and high concentrations of hydrogen sulfide (up to 3 mg/L). Stable isotope analysis of d18O of the oxic, chemocline, and anoxic layers indicate that these layers are likely from the Upper Floridan Aquifer. We applied quantitative polymerase chain reaction (q-PCR) and length-heterogeneity polymerase chain reaction (LH-PCR) to the four layers in order to characterize the microbial communities in terms of abundance. For bacteria, the surface layer had the highest relative abundance; the chemocline layer had the highest for archaea. All layers had similar relative abundance for eukarya. Through multidimensional scaling analysis, it was determined that the microbial communities are distinct within each layer and change temporally. For bacteria, relative abundance nearly doubled within three months in the oxic, chemocline, and anoxic layers. Similarly, archaea increased within the same three months within the oxic and chemocline layers, but decreased within the anoxic layer. No clear pattern for eukarya could be determined. These results begin to explain the ecosystem function of these kinds of sinks and will help guide better water management strategies in the future.