Microbial eukaryotes play critical roles in maintaining the health and stability of lake ecosystems. Consequently, investigating the spatial and temporal evolution of microbial eukaryotic community structures and associated environmental drivers can inform water ecology studies and water environmental protection efforts. In this study, sediment core samples were collected from Lakes Chaohu, Erhai, Lugu and Fuxian in China to reconstruct lake microbial eukaryotic community succession using sediment DNA profiling methods. Mantel tests were used to identify the key factors influencing community succession and explore the mechanisms underlying ecological network changes. Statistical analyses revealed that community structures and diversity in Lakes Chaohu and Erhai shifted in the 2000s and 1980s, respectively, while the communities of Lakes Fuxian and Lugu did not exhibit significant changes during the study period, which may be due to the oligotrophic state of these two lakes. Ciliophora and Chlorophyta were the primary eukaryotic microbial taxa in the four lakes during the study period, with overall relative abundances of 27.2 % and 22.3 %, respectively. The diversities of microbial eukaryotic communities in the lakes were significantly correlated with nutrient enrichment and meteorological factors. Lastly, the ecological niche gap of microbial eukaryotes gradually overlapped after community shifts in Lake Chaohu, while biased interactions strengthened. Overall, this study reveals the impacts of long-term human activities and climate change on lake ecosystems from multiple perspectives of microbial eukaryotic community structure, diversity and ecological network, these findings will contribute to integrate microbial indicators in freshwater environmental monitoring in the future.