In this study, a multiple-marker metabarcoding and high-throughput sequencing approach was applied to investigate the plankton diversity present in the Baltic Sea from winter to autumn. Four different markers targeting the 16S, 18S, and 28S ribosomal RNA genes were employed, including a dinoflagellate-specific marker. Typical seasonal changes were observed in phyto- and bacterioplankton communities. In phytoplankton, the appearance patterns of selected species followed the patterns also confirmed based on 20 years of phytoplankton monitoring data. In the case of zooplankton, no seasonal patterns were detected. For example, the majority of microzooplankton species were present throughout the sampling season. In total, 15 and 2 new zoo- and phytoplankton species were detected from the Baltic Sea. Metabarcoding and HTS approach was especially useful for detecting microzooplankton species, currently not included in the routine monitoring as well as investigating co-occurrence of different taxa. The results of this study further exemplify the efficiency of the molecular approach for biodiversity monitoring and the advantage of employing multiple markers through the detection of species not identifiable based on a single marker survey and/or by light microscopy.