Aquatic microeukaryotes (protists) provide essential ecosystem services from primary production to decomposition. The intermittent flows of non-perennial streams produce dynamics that shape the aquatic community and can alter these communities and ecological processes. Additionally, urban land use can amplify changes in flow regime and nutrient dynamics, thus bearing additional pressures that foster unique community structures that may affect stream health. Still, knowledge on microeukaryote responses to changes in land use and flow permanence is limited. This study aims to illuminate whether non-perennial streams host unique microeukaryotic diversity and whether urban land use modifies any flow effects. To address this goal, we collected microbial DNA from surface waters of perennial (n =15) and non-perennial (n =14) headwater streams in the East Fork Poplar Creek (EFPC) watershed in Oak Ridge, TN, during August 2023. EFPC flows along an urban to forested gradient, ranging from 61% to 0% impervious surface cover. We generated 18S rRNA gene amplicon libraries and analyzed the diversity and composition of the resulting microeukaryote sequence variants. Shannon diversity and evenness were lower in increasingly urbanized watersheds (respectively: rs = – 0.85, p < 0.001; rs = -0.43, p < 0.001) independent of flow regime. Flow regime and land use together explained 22% of the variance in community composition (PERMANOVA, p < 0.001), such that urban land use increased the community dissimilarity between perennial and non-perennial streams. Ciliophora (ciliates) dominated at non-perennial sites, whereas Chlorophyta (green algae) and diatoms were more prevalent in perennial locations. This study is part of a larger collaborative project investigating the dynamics of non-perennial and urban watersheds, and future work will link microbiome patterns to stream metabolic processes. Given the accelerating pace of urbanization and the reduction of stream flows worldwide, studies like this are crucial for understanding and managing the health of our streams.