The water spider (Argyroneta aquatic) is the only known spider to live a fully aquatic life. Therefore, it has been the subject of a series of studies on various aspects of its unique biology such as its reproductive behavior, sexual dimorphism, physiology, genetics, and silk. However, there have been relatively few studies on the hydroecology of where water spiders live. The water spider habitat in Eundae-ri, Yeoncheon is the only habitat for A. aquatica, a globally rare species, in South Korea. In this region, the water level of the wetland is automatically adjusted to groundwater owing to continued drying. Here, the surface water, wetland, and groundwater near the A. aquatica habitat were studied using hydrochemical, microbiological, and correlation analyses. The hydrochemical properties—water temperature, pH, electrical conductivity, dissolved oxygen (DO), oxidation reduction potential, and turbidity—of the surface water and wetland were similar. The Piper diagrams revealed that the wetlands, surface water, and most of the groundwater portrayed Ca-HCO3-type properties, whereas only areas where the water level of the wetland was controlled displayed Na-HCO3-type properties. Furthermore, the NO3 content was too low to be detected in the wetland, indicating clean and non-polluted water conditions; additionally, heavier oxygen-hydrogen isotopes were observed because these regions were climatically affected by the wetland. The dominant bacteria were Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Verrucomicrobia, and Nitrospirae. The correlation analysis revealed that the major environmental control factors of the A. aquatica habitat were DO, temperature, and pH, and the related bacteria were Cyanobacteria, Actinobacteria, and Verrucomicrobia.