Remarkably, unlike other parts of Europe, the ecology of mayflies in the southeastern regions is still poorly known. Here we present the first comprehensive study of Ephemeroptera in the tufa-depositing habitats of the Dinaric Karst. The study was conducted in Plitvice Lakes National Park monthly during a one-year period (2007–2008) in different types of habitats (springs, streams, mountainous rivers, tufa barriers). The aims of the study were to determine mayfly composition, abundance, spatial distribution and habitat preferences, and to examine the environmental factors important for the structuring of mayfly assemblages in Plitvice Lakes National Park. The mayfly fauna of tufa-depositing habitats was composed of 14 species (20 taxa). Water temperature, pH and ammonium concentration were the most important environmental variables explaining mayfly assemblages. Mayfly assemblages grouped according to habitat type. Generally, the most favourable habitat type was mountainous stream, tufa barriers were less favourable, and the least favourable were springs. Our results confirmed that mayflies are a powerful tool as descriptors of their environment, as the presence or absence of certain mayflies was strongly influenced by physico-chemical water properties.