The pollution of karst aquifers by acid mine drainage (AMD) waters is increasing. Major and minor ions (Ca2+, Mg2+, HCO3−, SO42−, F−, and Fe), stable sulfur and oxygen isotopes of dissolved sulfates (δ34SSO4 and δ18OSO4) and oxygen isotope of water (δ18OH2O), were analyzed in rainwater, surface water, groundwater, and AMD water sampled from the Babu subterranean stream watershed, in Southwest China. The principal aim of this study was to explore the impact of AMD waters on the evolution of karst aquifers. Based on hydrogeochemistry and stable isotopes (δ18OH2O, δ18OSO4 and δ34SSO4): (1) the chemistry of AMD waters was primarily controlled by pyrite oxidation, karst conduit water by AMD waters and mixing with calcite and dolomite dissolution, and spring water by atmospheric precipitation and carbonate dissolution; (2) contamination of the karst conduit water was mainly attributed to the input of AMD waters, resulting in a shift of δ34SSO4 towards more negative values (from 3.4‰ to −13.2‰); (3) the quality of karst conduit water changed from suitable to unsuitable for irrigation and drinking, particularly due to the increase in total Fe, SO42−, and F− concentrations, reflecting the cumulative effect of AMD waters derived from tailings dumps; this influence was enhanced during rainstorm/drought and anthropogenic activities; and (4) the flow of contaminated groundwater through the conduit promoted the dissolution of carbonates, especially during the dry season due to the greater proportion of AMD in the groundwater. This released more CO2 to the atmosphere. We believe that analysis of stable isotopes (δ18OH2O, δ18OSO4, and δ34SSO4), combined with hydrogeochemistry, is effective for exploring the impact of AMD on karst aquifers. Therefore, reasonable treatment methods should be taken to reduce the negative impacts of tailings dumps on karst aquifers.