The composition of phytoplankton community is the basis for environmental monitoring and assessment of the ecological status of aquatic ecosystems. Community composition studies of phytoplankton have been based on time-consuming and expertise-demanding light microscopy analyses. Molecular methods have the potential to replace microscopy, but the high copy number variation of ribosomal genes and the lack of universal primers for simultaneous amplification of prokaryotic and eukaryotic genes complicate data interpretation. In this study, we used our previously developed directional primer-independent high-throughput sequencing (HTS) approach to analyze 16S and 18S rRNA community structures. Comparison of 83 boreal lake samples showed that the relative abundances of eukaryotic phytoplankton at class level and prokaryotic cyanobacteria at order level were consistent between HTS and microscopy results. At the genus level, the results had low correspondence, mainly due to lack of sequences in the reference library. HTS was superior to identify genera that are extensively represented in the reference databases but lack specific morphological characteristics. Targeted metatranscriptomics proved to be a feasible method to complement the microscopy analysis. The metatranscriptomics can also be applied without linking the sequences to taxonomy. However, direct indexing of the sequences to their environmental indicator values needs collections of more comprehensive sample sets, as long as the coverage of molecular barcodes of eukaryotic species remains insufficient.