Phosphorus enrichment of stream ecosystems generally increases primary production in the benthos, but the consequences of eutrophication for the nutritional quality of periphyton for grazers are less clear. On short timescales, high phosphorus inputs may lead to reduced C:P ratios and high essential fatty acid contents of periphyton, which are both considered important determinants of food quality for grazers. However, nutrient enrichment may alter the taxonomic composition of periphyton and favor the growth of less palatable algal taxa. In this study, periphyton was grown under a gradient of dissolved phosphorus availability from 5 to 100 µg P · L⁻¹, to investigate eutrophication effects on periphyton taxonomy, C:N:P stoichiometry, and fatty acid composition. After 1 month, periphyton grown under oligotrophic conditions was mainly composed of diatoms (~86%). With increasing phosphorus availability, diatoms were gradually outcompeted by chlorophytes and cyanobacteria, which were the predominant taxon under eutrophic conditions. Unexpectedly, periphyton C:P ratios increased with greater phosphorus supply, from ~280 under oligotrophic conditions up to ~790 at 100 µg · L⁻¹, reflecting a tendency of chlorophytes and cyanobacteria to produce more biomass per unit of assimilated phosphorus compared to diatoms. Periphyton content of essential polyunsaturated fatty acids relative to biomass followed a unimodal relationship with phosphorus availability and peaked at intermediate phosphorus levels, likely as a result of both taxonomic and nutrient effects. Our results demonstrate that phosphorus-driven eutrophication of freshwater ecosystems may worsen periphyton nutritional quality due to taxonomic sorting, which may further lead to lower growth and reproduction of herbivores.