To effectively address biodiversity loss, it is essential to prioritize conservation efforts by identifying areas of high conservation value. Ecological uniqueness is a valuable metric for this purpose that decomposes beta diversity into local contributions to beta diversity (LCBD), thereby measuring the contribution of each site within a region to overall biodiversity variation. LCBD has been used extensively to evaluate ecological uniqueness from community composition data, but biodiversity is a multifaceted concept, and community-based ecological uniqueness may not capture the full range of ecological uniqueness occurring at other levels of biological organization. We investigated ecological uniqueness estimates derived from community and species population levels in a watershed of south-central Chile and analyzed their responses to water and habitat quality variables. Ecological uniqueness was estimated at the community level from fish and macroinvertebrate assemblages and at the population level using genetic and morphological data gathered for two invertebrates, the gastropod Chilina dombeiana and the water bug Aquarius chilensis. Our results revealed low spatial congruence between the levels of ecological uniqueness calculated for these different biodiversity components, with mismatches occurring among sites with high LCBD values. Water and habitat quality were major drivers of beta diversity in this watershed, accounting for 43.8% to 74.3% of the spatial variation in LCBDs, and their effects differed among the ecological uniqueness estimates. Overall, our results underscore the idiosyncratic nature of ecological uniqueness metrics, emphasizing the importance of using multiple components of biodiversity to guide conservation actions.