Caves serve as exceptional ecological models for studying the environmental factors that shape biological communities. However, there has been limited exploration into understanding how these environmental factors affect communities' distribution and responses to habitat traits within the same cave system. To address this gap, we employed standardized sampling methodologies across two scales within a limestone cave located in the Brazilian semi-arid region (Pedro Cassiano cave). Our objective was to uncover how substrate characteristics and conditions may contribute to variations in the composition and richness of invertebrates (troglobitic and nontroglobitic). Furthermore, we aimed to comprehend the habitat selection of some troglobitic species. This particular cave hosts a rich fauna, with 21 obligate cave species (20 invertebrates and 1 fish), which positions it as new Hotspot of Subterranean Biodiversity (term used for caves that have 20 or more troglobitic species) in South America. Pedro Cassiano Cave has two separate tributary drainages that flow from its final section and merge into a single drainage system that exits through the cave entrance (resurgence). We observed that the distribution of non-troglobitic invertebrate richness differed when each tributary branch was analyzed independently, indicating that the distribution patterns of subterranean species can be complex. Microhabitat characteristics such as heterogeneity, the amount of sand, and the availability of trophic resources explained a significant portion of the invertebrate community distribution in this cave, although this influence was scaledependent. Finally, we emphasized the urgency of conservation efforts for this important cave.