Metabarcoding is a time-saving and cost-effective approach that promises to overcome issues associated with traditional plankton taxonomy (i.e. lack of specialized personnel, time-consuming methodologies, difficulties in assignment of larval stages, and detection of cryptic species). In this regard, we applied metabarcoding using the18S rDNA V9 region to samples collected throughout 1 yr from the Estuary of Bilbao (Basque Country, Spain) in order to characterize the annual cycle of the eukaryotic plankton community. We found clear patterns of spatial and seasonal environmental variability that drive the distribution and abundance of the plankton assemblage throughout the year, thus confirming results of previous studies using microscopic identification of the planktonic species. Our results also suggest that the low oxygen period during summer in the inner part of the estuary (salinity 30) and the thermal variation from winter to summer are among the main environmental drivers of the plankton community of the Estuary of Bilbao. Finally, we report misidentification of some species (e.g. Cyclopina gracilis, Maristentor dinoferus), which highlights the need for more comprehensive reference sequence databases in order to overcome this limitation of metabarcoding.