For over five decades, the biodiversity of freshwater sponges from Yucatan cenotes has remained largely unexplored. Scientific documentation is limited to studies from 1936 and 1968, which highlights a significant knowledge gap. It is here reported the presence of the freshwater sponge Ephydatia fluviatilis in the Sabak-Ha and Suhem cenotes of Yucatan, Mexico. It is provided detailed descriptions of the spicules to accurately characterize morphology and ease comparison with other Ephydatia species. Additionally, we conducted molecular analyses using DNA sequences from the ITS1, 5.8S, and ITS2 regions, as well as partial 18S and 28S sequences, to determine phylogenetic relationships within the Spongillidae family. DNA sequences from both cenotes were nearly identical to E. fluviatilis GenBank sequences (<3% divergence), confirming species identification. Spicule analysis revealed gemmuloscleres birotules with shafts densely covered by spines as long as the rotular rays as recorded throughout the entire geographic range of this species in both lentic and lotic water bodies. This study expands our knowledge of the diversity and distribution of freshwater sponges in Yucatan cenotes, where only Spongilla cenota and Radiospongilla crateriformis had been previously reported. Our results lay the groundwork for future research on cenote sponge biodiversity in this unique karst ecosystem and underscore the importance of integrating molecular and morphological data for accurate species identification.