Secondary production in freshwater zooplankton is frequently limited by the food quality of phytoplankton. One important parameter of phytoplankton food quality are essential polyunsaturated fatty acids (PUFAs). Since the fatty acid composition of phytoplankton is variable and depends on the algae's nutrient supply status, inorganic nutrient supply may affect the algal PUFA composition. Therefore, an indirect transfer of the effects of nutrient availability on zooplankton by changes in algal PUFA composition is conceivable. While the phosphorus (P) supply in lakes is largely decreasing, nitrogen (N) inputs continue to increase. This paper presents data from a mesocosm field experiment in which we exposed phytoplankton communities to increasing N enrichment. As a consequence, the PUFA composition of the phytoplankton community changed. With increasing nitrogen fertilisation, we observed lower quantities of essential PUFAs, together with a decrease in the abundances of the dominant herbivorous zooplankton Daphnia sp. Their biomass was significantly correlated with phytoplankton PUFA content (C18:3 ω3, C20:5 ω3, C18:2 ω6). Our data therefore indicate that changes in nitrogen supply, together with the resultant changes in phytoplankton food quality, can negatively affect the secondary production of herbivorous zooplankton by reducing the availability of essential polyunsaturated fatty acids.