Caves are unique environments that can support ecosystems largely independent of photosynthetic primary production and are heavily influence by the geochemistry of the geologic units they are formed within. This study investigated five epigenic cave systems in the Black Hills of South Dakota—Rushmore Cave, Bethlehem Cave, Stagebarn Cave, Dahm Springs Cave, and Brooks Cave—with the goal of delimiting microbial habitability. Habitability, defined as the potential for life to thrive or survive, was determined by examining the physical, biological, and geochemical composition of waters and sediments within these five caves. Microbial (aerobic) respiration rates and DNA concentrations in cave sediments were used to define biological activity. Microbial respiration rates within sediments were found to strongly correlate with organic carbon content (p-value = 0.004) through Spearman Rho tests but did not significantly correlate with DNA concentration. The correlation between microbial respiration rate and organic carbon content can be attributed to heterotrophic activity. However, for chemoautotrophic bacteria in caves to gain energy though biomineralization of CaCO3 from inorganic carbon, cave waters should be supersaturated with respect to CaCO3. Every pool that was sampled in this study was supersaturated with respect to calcite and aragonite, increasing the potential habitability for chemoautotrophs. Trace metal data also provide important constraints on active metabolisms potentially in each cave, such as ions with a valency of +2 (i.e., Fe2+, Mn2+, Ba2+, and Sr2+) and the precipitation of metal-bound carbonate minerals siderite (FeCO3), rhodochrosite (MnCO3), witherite (BaCO3), and strontianite (SrCO3). Using non-metric multidimensional scaling, each cave was found to have distinct geochemistry despite occurring in the same geologic unit, the Pahasapa Formation. Some of the most important distinguishing parameters of the caves were sediment organic and inorganic carbon content, dissolved organic (TOC) and inorganic (TIC) carbon in pool water, air CO2 concentration, and various dissolved ion concentrations. Distance between caves did not play a major role in subsurface environmental variability. Surface sediment chemistry and microbial activity above each cave did not correlate with subsurface sediments within each cave. Tourism, however, was found to heavily impact the cave environment in Rushmore Cave through an increased CO2 concentration from visitor respiration, introduction of dissolved metals from pollutants (coins) in pools, and elevated NO3-. The results of this study provide important insights into the potential habitability of Black Hills caves by microorganisms. Additionally, these results provide context for future studies of microbial diversity within the region, and for exploring the limits to life in nutrient-limited ecosystems.