Microbial eukaryotes play a vital role in biogeochemical cycles and aquatic food webs. Over past decades, their taxonomic diversity has been investigated using short-read DNA metabarcoding, which, while effective, is hampered by the limited resolution of the targeted regions. In this study, we utilised both short-read (Illumina) and long-read (PacBio) metabarcoding approaches to analyse eukaryotic diversity in a lake ecosystem over one year, allowing for a direct comparison of the results obtained from each method. Our metabarcoding analysis revealed a high degree of congruence between long-read and short-read data at broad taxonomic levels. However, at finer taxonomic scales, such as the genus level, long-read sequencing achieved higher resolution, enabling more precise identification of microbial eukaryotes. This enhanced taxonomic resolution proved especially valuable for tracking seasonal dynamics within key groups, including ciliates and chytrid fungi. The resolution offered by long-read sequencing provides a more detailed picture of the microbial eukaryotic community, thereby facilitating the exploration of ecological interactions at a finer scale. For example, this approach allowed us to monitor the dynamics of the chytrid genus Zygorhizidium in relation to various diatom genera, which are primary targets for chytrid parasitism.