Marine microbial flagellates form an important part of marine ecosystems, and play an essential role in maintaining ecosystem functions. However, the underlying biogeographic processes and ecological effects that shape marine microbial flagellate communities (MFCs) on the geographical scale (~ 2,000 km) remain unclear, especially how their composition is related to movements of water masses. In this study, high-throughput sequencing of 18S rRNA genes was conducted to survey two size-fractioned groups (0.8–2.0 μm for pico-sized and 2.0–20 μm for nano-sized groups) of MFCs in three subtropic-tropic marginal seas of China. Furthermore, the impacts of environmental factors, spatial factors, and water masses on MFCs were explored and compared across different spatiotemporal conditions. The results demonstrate non-random biogeographic distributions of MFCs in the studied area. These distributions were affected by several ecological processes, such as environmental selection, dispersal limitation, neutral process, and interactions within communities. These processes were driven by complex water masses that formed on a geographical scale. Notably, environmental heterogeneity was identified as the principal determinant of MFCs in each sea area. However, the importance of spatial factors increased with the spatial scale, which weakened biotic interactions within the community on a geographical scale. This effect was more apparent in nano-sized MFCs, indicating stronger dispersal limitation because of their larger cells and weaker dispersal ability. In summary, this study expands the available knowledge on the dynamic biogeographic patterns of MFCs associated with water masses on a geographical scale where strong spatial and environmental gradients exist.