Here we report a late Holocene fossil-rich cave deposit from Cueva de los Nesofontes, Mayabeque Province, Cuba. The deposit's formation and its fauna were studied through a multidisciplinary approach that included stable isotope analyses, radiocarbon chronology, and stratigraphy. Thousands of microvertebrate skeletal remains were recovered, representing a diverse land vertebrate fauna that included threatened and extinct species. The deposit is characterized by profuse Nesophontes remains due to raptor predation. Previously unreported last appearance dates (LADs) are provided for the extinct 'island-shrew' Nesophontes major, the bats Artibeus anthonyi, and Phyllops vetus. Radiocarbon (14 C AMS) age estimates between ~1960 rcyr BP and the present were acquired for the assemblage. The presence of locally extinct species, including the endemic parakeet Psittacara eups, the flicker Colaptes cf. fernandinae, the bat Antrozous koopmani, and the eulipotyphlan Solenodon cubanus, suggests that these species had broader distributions in the near past. Isotope analyses and faunal composition indicate the previous presence of diverse habitats, including palm grove savannas and mixed woodlands near the cave. Isotopes also provide insight into the habitat and coexistence of the extinct bat Artibeus anthonyi and extant A. jamaicensis, the diet of Nesophontes major, and local paleoenvironmental conditions. Oxygen isotopes reveal an excursion suggestive of drier/colder local conditions between 660 and 770 AD. Our research further expands the understanding of Cuban Quaternary extinction episodes and provides data on the distribution and paleoecology of extinct taxa. It supports the conclusion that many Cuban extinct species survived well into the pre-Columbian late Holocene and retained wide distribution ranges until European colonization.