Despite the recent increase in the number of mayfly studies in karst freshwater habitats, their biology and ecology in springs are still poorly characterized. Therefore, we studied mayfly assemblages in a European karst rheocrene spring at five microhabitats monthly over a one-year period. Three species were recorded: Baetis alpinus (Pictet, 1843), Baetis rhodani (Pictet, 1843) and Rhithrogena braaschi (Jacob, 1974). The latter species represents a new record for the fauna of Bosnia and Herzegovina. All three species inhabited all studied microhabitats but with varying abundance. Individual species were associated with a specific substrate type and/or water velocity and/or water depth. The grazer/scraper Rh. braaschi was most common at microhabitats with inorganic substrate (cobbles, mixture of pebbles and sand), moderate water velocity and higher water depth. The rheophilic grazer/scraper and gatherer/collector B. alpinus was most common at microhabitats with mosses and highest water velocity. The grazer/scraper and gatherer/collector B. rhodani was recorded at all microhabitats, yet due to its preference for moderate water velocity, the highest number of individuals were collected from cobbles. We recorded movements of mayfly nymphs among the available microhabitats during their life cycles, due likely to their dietary requirements and search for suitable refugia. Baetis alpinus has a bivoltine, B. rhodani polyvoltine and Rh. braaschi univoltine life cycle with a long emergence period. The results presented here contribute to the knowledge of spring and mayfly ecology. Abbreviations: HCA-Hierarchical cluster analysis, NMDS-Non-metric multidimensional scaling, UPGMA (Unweighted Pair Group Method with Arithmetic Mean). Nomenclature: Bauernfeind and Soldán (2012). Mayfly ecological traits 249 their biota remain insufficiently investigated and a potential and interesting subject of research.