Aquatic Empididae (Diptera) in the subfamilies Hemerodromiinae and Clinocerinae were used as a model to provide the first appraisal of diversity, endemism, environmental preferences and anthropic threats to Diptera communities of calcareous streams and tufa ecosystems of tropical karst habitats of Thailand. Hemerodromia Mg. (Hemerodromiinae) was abundant and speciose with 17 spp., of which six were associated with tufa, two with calcareous streams, three occurred on both tufa and streams and six were eurytopic species also found away from karst. Clinocerinae were only represented by one rare species of Clinocera Mg. Local and regional‐scale endemism was apparent at species level in Hemerodromia. Assemblage similarity in tufa habitats decayed with distance but cluster analysis provided only weak support for community‐level endemism at any geographic scale. Ordination indicated that benthic substrate and canopy cover were the most important environmental variables for many species. The six obligate tufa species of Hemerodromia were absolutely associated with rapid shallow flows of water over tufa surfaces at waterfalls. Species richness and abundance varied through the monsoon cycle but there was little evidence of simple correlations with seasonal changes in water temperature, conductivity and pH. The hypothesis that cold‐adapted aquatic Empididae might have been marooned in cold emergent groundwaters at lower elevations was not supported. It is concluded that tropical tufa ecosystems in Thailand support distinctive, rich and often endemic populations of stenotopic species of considerable biological and conservation interest. Deteriorative changes linked to tourism, water abstraction and agricultural activity could threaten these unique communities.