Sterols are essential dietary nutrients for most aquatic invertebrates and an inadequate dietary sterol supply can adversely influence various life history traits. The sterol composition of microalgae is highly diverse, which may have consequences for zooplankton nutrition because sterols differ in their nutritional quality. We investigated the sterol content and composition of a number of eukaryotic freshwater microalgae belonging to different classes. The aim was to increase our knowledge of sterol profiles and biomass-related sterol concentrations in freshwater algae and to reveal sterol-mediated nutritional constraints that consumers potentially have to cope with. Our data suggest that zooplankton consumers are potentially confronted with low dietary sterol concentrations, depending on the phytoplankton species composition. Total sterol concentrations were found to be low especially in representatives of the genera Chlamydomonas, Choricystis and Gomphonema. Moreover, our findings suggest that the predominance of certain chlorophytes (Scenedesmus, Ankistrodesmus and Monoraphidium) in phytoplankton assemblages may cause sterol metabolic constraints in consumers because of high dietary concentrations of nutritionally inadequate Δ⁷ and Δ^7,22 sterols. The sterol profiles we present here can serve as reference profiles for particular algae to assess their biochemical food quality for freshwater consumers and provide a first step toward assessing the significance of sterol-mediated nutritional constraints under field conditions.