The sulfuric acid speleogenesis (SAS) model was introduced in the early 1970s from observations of Lower Kane Cave, Wyoming, and was proposed as a cave-enlargement process due to primarily H2S autoxidation to sulfuric acid and subaerial replacement of carbonate by gypsum. Here we present a reexamination of the SAS type locality in which we make use of uniquely applied geochemical and microbiological methods. Little H2S escapes to the cave atmosphere, or is lost by abiotic autoxidation, and instead the primary H2S loss mechanism is by subaqueous sulfur-oxidizing bacterial communities that consume H2S. Filamentous “Epsilonproteobacteria” and Gammaproteobacteria, characterized by fluorescence in situ hybridization, colonize carbonate surfaces and generate sulfuric acid as a metabolic byproduct. The bacteria focus carbonate dissolution by locally depressing pH, compared to bulk cave waters near equilibrium or slightly supersaturated with calcite. These findings show that SAS occurs in subaqueous environments and potentially at much greater phreatic depths in carbonate aquifers, thereby offering new insights into the microbial roles in subsurface karstification.