AbstractThe advent of high‐throughput sequencing has led to the discovery of a considerable diversity of microbial eukaryotes in aquatic ecosystems, nevertheless, their function and contribution to the trophic food web functioning remain poorly characterized especially in freshwater ecosystems. Based on metabarcoding data obtained from a meromictic lake ecosystem (Pavin, France), we performed a morpho‐physio‐phenological traits‐based approach to infer functional groups of microbial eukaryotes. Metatranscriptomic data were also analysed to assess the metabolic potential of these groups across the diel cycle, size fraction, sampling depth, and periods. Our analysis highlights a huge microbial eukaryotic diversity in the monimolimnion characterized by numerous saprotrophs expressing transcripts related to sulfur and nitrate metabolism as well as dissolved and particulate organic matter degradation. We also describe strong seasonal variations of microbial eukaryotes in the mixolimnion, especially for parasites and mixoplankton. It appears that the water mixing (occurring during spring and autumn) which benefits photosynthetic host communities also promotes parasitic fungi dissemination and over‐expression of genes involved in the zoospore phototaxis and stage transition in the parasitic cycle. Mixoplanktonic haptophytes over‐expressing photosynthesis‐, endocytosis‐ and phagosome‐linked genes under nutrient limitation also suggest that phagotrophy may provide them an advantage over non‐phagotrophic phytoplankton.