We studied benthic assemblages through X-Batún, a continental freshwater cenote and its associated submerged cave located in San Antonio Mulix (Yucatán, Mexico). Using cave diving techniques, we collected sediment samples at four zones of the system. We extracted and counted individuals of benthic species in three replicates of 5 grams of wet sediment at each site. The biological composition was integrated by 15 species from eight higher taxonomic groups. Non-metric multidimensional scaling distinguished four assemblages that coincided with surface, open water, cavern and cave zones. ANOSIM test revealed significant differences between the assemblages. In the deeper zones of the cenote characterized by twilight and total darkness, Ostracoda and Gastropoda show the highest diversity and abundance, with practical absence in surface sediments. This pattern may suggest ecological interactions with chemosynthetic bacterial activity. Surface shows an assemblage typical of epigean environments. Environmental variables along the cenote varied little from the upper layers to bottom. Linear correlation and detrended canonical analysis revealed that light is the main driver of benthic species assemblages. Temperature, pH, and dissolved oxygen exert higher influence at individual biological benthic assemblage in X-Batún.