Eutrophication (as an increase in total phosphorus [TP]) increases harmful algal blooms and reduces the proportion of high-quality phytoplankton in seston and the content of ω-3 long-chain polyunsaturated fatty acids (eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) in fish. However, it is not well-known how eutrophication affects the overall nutritional value of phytoplankton. Therefore, we studied the impact of eutrophication on the production (as concentration; μg L−1) and content (μg mg C−1) of amino acids, EPA, DHA, and sterols, i.e., the nutritional value of phytoplankton in 107 boreal lakes. The lakes were categorized in seven TP concentration categories ranging from ultra-oligotrophic (<5 μg L−1) to highly eutrophic (>50 μg L−1). Phytoplankton total biomass increased with TP as expected, but in contrast to previous studies, the contribution of high-quality phytoplankton did not decrease with TP. However, the high variation reflected instability in the phytoplankton community structure in eutrophic lakes. We found that the concentration of amino acids increased in the epilimnion whereas the concentration of sterols decreased with increasing TP. In terms of phytoplankton nutritional value, amino acids, EPA, DHA, and sterols showed a significant quadratic relationship with the lake trophic status. More specifically, the amino acid contents were the same in the oligo- and mesotrophic lakes, but substantially lower in the eutrophic lakes (TP > 35 μg L−1/1.13 μmol L−1). The highest EPA and DHA content in phytoplankton was found in the mesotrophic lakes, whereas the sterol content was highest in the oligotrophic lakes. Based on these results, the nutritional value of phytoplankton reduces with eutrophication, although the contribution of high-quality algae does not decrease. Therefore, the results emphasize that eutrophication, as excess TP, reduces the nutritional value of phytoplankton, which may have a significant impact on the nutritional value of zooplankton, fish, and other aquatic animals at higher food web levels.