The karst underground river ecosystem of Yucatan peninsula is composed of cave systems and sinkholes. The microbial diversity of water from this underground river has been studied, but, structure of the microbial community in its cave sediments remained largely unknown. Here we describe how the microbial community structure of these sediments changes due to different environmental conditions found in sediment zones along the caves of a coastal and an inland sinkhole. We found that dominant microbial groups varied according to the type of sinkhole (Coastal: Chloroflexi and Crenarchaeota; inland: Methylomirabilota and Acidobacteriota) and that the community structures differed both among sinkhole types, and within the sediment zones that were studied. These microorganisms are associated with different types of metabolism, and differed from a microbial community dominated by sulfate reducers at the coastal sinkhole, to one dominated by methylotrophs at the inland sinkhole, suggesting there are biogeochemical processes in the coastal and inland sinkholes that lead to changes in the microbial composition of the underground river ecosystem’s sediments. Our results suggest sediments from unexplored sinkhole caves are unique environmental niches with distinct microbial assemblages that putatively play an important role in the biogeochemical cycles of these ecosystems.