Functional diversity is a key component of biodiversity that reflects various dimensions of ecosystem functioning and the roles organisms play within communities and ecosystems. It is widely used to understand how ecological processes influence biotic assemblages. With an aim to increase our knowledge about dragonfly ecological requirements in tufa-depositing karst habitats, we assessed functional diversity of their assemblages, various life history traits (e.g., stream zonation preference, substrate preference, reproduction type), and relationship between functional diversity and physico-chemical water properties in three types of karst lotic habitats (springs, streams, and tufa barriers) in a biodiversity hotspot in the western Balkan Peninsula. Dragonfly functional diversity was mainly characterized by traits typical for lotic rheophile species with medium dispersal capacity. Among the investigated habitats, tufa barriers, characterized by higher (micro)habitat heterogeneity, higher water velocity, as well as lower conductivity and concentration of nitrates, can be considered as dragonfly functional diversity hotspots. Functional diversity and most of the life history traits were comparable among different substrate types in the studied habitats, indicating higher importance of habitat type in shaping dragonfly functional diversity patterns in karst lotic habitats. Our results should be considered in the management and conservation activities of vulnerable karst freshwater ecosystems and their dragonfly assemblages.