Chemical gradients between fresh, brackish and saline waters shape biogeochemical reactions and organic matter transformation within subterranean estuaries. In the Yucatán Peninsula’s karst subterranean estuary (KSE), methane and dissolved organic matter generated during the anaerobic decomposition of tropical forest vegetation are transported into flooded cave networks where microbial consumption greatly reduces their concentrations in the groundwater. To test the hypothesis that chemoclines associated with salinity gradients of the KSE are sites of methane oxidation, we obtained methane concentration and δ¹³C profiles of unprecedented vertical resolution from within a fully-submerged cave system located 6.6 km inland from the coastline using the ‘OctoPiPi’ (OPP) water sampler. Along a 12–24 cm thick low-salinity-halocline at ∼4.5 m water depth, salinity increased from fresh to brackish (0.2–1.8 psu), methane concentrations decreased, and δ¹³C values increased, as expected for microbial methane oxidation. The underlying brackish water had elevated oxygen concentrations compared to the always anoxic freshwater, suggesting that aerobic methane oxidation is the dominant process facilitating methane consumption. By contrast, as salinity increased from 1.8 to 36 psu through a 24–36 cm thick high-salinity-halocline between the meteoric lens and the saline groundwater at ∼20 m water depth, methane concentrations and δ¹³C values were constant. Conservative mixing and kinetic isotope models incorporating the methane data confirm a hotspot for microbial methane oxidation at the low-salinity-halocline. At least 98% of methane originating in the anoxic freshwaters was removed before its transport via channelized flow towards the coastline. These findings provide novel insight into the spatial constraints of methane dynamics within a karst subterranean estuary.