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The impacts of environmental change on Lake Biwa have been explored for decades, with water monitoring and palaeolimnological studies revealing how environmental forcing, including climate warming, eutrophication, water level manipulation and human manipulation of fish populations, has influenced the food web of Lake Biwa. However, these studies have rarely accounted for microbial food‐web components. This knowledge gap is mostly due to the lack of time series spanning more than a couple of decades, coupled with the high taxonomical expertise required to identify organisms belonging to very diverse groups.The use of a sedimentary DNA approach allows for the reconstruction of past changes in the diversity, composition and structure of the microbial eukaryotic community of aquatic systems. The application of 18S metabarcoding has been proven successful to describe the response of unicellular eukaryotes (protists) and aquatic fungi in lake ecosystems, encompassing a large taxonomic and functional diversity such as phototrophs, heterotrophs and mixotrophs.We applied 18S metabarcoding to 31 sediment core samples from Lake Biwa, spanning the past 100 years and explored the response of microbial eukaryotic communities to changes in multiple environmental stressors, including nutrient levels, lake water level, climate, as well as fish and zooplankton biomass for the period 1973–2010.We found that the manipulation of the water level and changes in fish community composition were the primary factors impacting (indirectly) the structure of the lake microbial eukaryotic community with minor, but significant, effects of climate warming and phosphorus levels. Co‐occurrence network analysis highlighted the potential food web impacts on the microbial eukaryotic community, suggesting that organisms from this compartment were impacted by both bottom‐up and top‐down processes. [ABSTRACT FROM AUTHOR]