Heterotrophic flagellates (HFs) are important members of the aquatic microbial food web. However, information on their spatial patterns in relation to eutrophication is limited. Here, we examined the composition and spatial distributions of HFs (<3 μm) in subtropical coastal waters of different trophic status by re-analyzing two previously published small subunit rDNA pyrosequence datasets using information from the newly launched Protist Ribosomal Reference database (PR²). Whereas the contributions of different major clades composing the Marine Stramenopiles (MASTs), picobiliphytes and Chrysophyceae were found relatively comparable between the stations, contrasting compositions of the Marine Alveolates (MALV) groups I and II were observed. The high and relatively stable contribution of MAST-1, -3 and -7 among the MASTs in both stations suggest their importance as bacterial grazers in coastal waters, irrespective of trophic status. By contrast, the dominance of clades 3, 5 and 14 of MALV II in the eutrophic station implies their importance in regulating the dinoflagellate population at the site. Our study provides insights into the ecological importance of different HF groups in eutrophic coastal ecosystems.