Submerged caves, locally referred to as cenotes, can be found throughout the Yucatan Peninsula of Mexico. These nutrient poor, aphotic “underground estuaries” lack photosynthetic primary productivity, but are often found underlying high primary productivity areas such as mangroves and tropical forests. Adjacent ecosystems contribute organic carbon to the cave systems via percolation, where it is then utilized by the obligate, cave-dwelling fish and invertebrates. Another potential pathway through which organic carbon can enter the cave food web is through chemosynthesis. Chemoautotrophic sulfur-oxidizing or nitrifying bacteria have been found in the hydrogen sulfide layer or in the sediments of some anchialine caves. Our study utilizes 13C/12C and 15N/14N stable isotopes as well as 14C radiocarbon dating to determine and compare the sources of organic matter entering a coastal anchialine cave (Cenote Aak Kimin) versus an inland cave (Cenote Maya Blue) in the Yucatan Peninsula. Stable isotopes have long been employed in tropic investigations. This study, however, is the first to utilize radiocarbon isotopes in anchialine caves. The use of both stable and radiocarbon isotopes as source indicators provides greater discrimination in systems that contain numerous carbon sources or indistinct trophic levels, particularly to distinguish between chemoautotrophic versus photosynthetically derived carbon. Results indicate that chemosynthetically derived organic carbon contributes substantially to the diet of some crustaceans, such as the stygobitic shrimp Typhlatya, while other species remain dependent on detrital inputs. Depleted δ13C values and aged radiocarbon values (as low as -47.51‰ and 1840 yrs. for Typhlatya spp.) in comparison to particulate and sediment δ13C values (lowest -32.07‰ and -28.43‰, respectively). A comparison of isotopic values between Cenote Aak Kimin and Cenote Maya Blue suggests that the trophic web of the coastal cave incorporates more photosynthetic or detrital carbon, while the inland cave, with more depleted 13C and 14C values, relies more heavily on chemoautotrophic carbon. Within both systems, however, distinct photosynthetic and chemoautotrophic levels were identified. Water quality parameters, especially dissolved oxygen and pH, support the hypothesis of bacterial activity at the halocline. Anchialine systems in the Yucatan Peninsula are threatened due to increases in tourism, development, and pollution. Quantifying and qualifying the inputs of organic carbon is vital for the management and conservation of the area’s freshwater resources.