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The Fern Cave System, developed in the western escarpment of the Southern Cumberland Plateau of the Interior Low Plateau karst region in Northeastern Alabama, USA, is a global hotspot of cave-limited biodiversity as well as home to the largest winter hibernaculum for the federally endangered Gray Bat (Myotis grisescens). We combined the existing literature, museum accessions, and database occurrences with new observations from bioinventory efforts conducted in 2018–2022 to generate an updated list of troglobiotic and stygobiotic species for the Fern Cave System. Our list of cave-limited fauna totals twenty-seven species, including nineteen troglobionts and eight stygobionts. Two pseudoscorpions are endemic to the Fern Cave System: Tyrannochthonius torodei and Alabamocreagris mortis. The exceptional diversity at Fern Cave is likely associated with several factors, such as the high dispersal potential of cave fauna associated with expansive karst exposures along the Southern Cumberland Plateau, high surface productivity, organic input from a large bat colony, favorable climate throughout the Pleistocene, and location within a larger regional hotspot of subterranean biodiversity. Nine species are of conservation concern, including the recently discovered Alabama cave shrimp Palaemonias alabamae, because of their small range sizes, few occurrences, and several potential threats.