Estuaries present a very dynamic environment for phytoplankton with large changes in salinity occurring over relatively short distances, usually with high turbidity. We investigated the photosynthetic characteristics of phytoplankton in the Scheldt estuary (Belgium and the Netherlands) along a salinity gradient using community and single-cell fluorescence measurements. Single-cell fluorescence measurements offer the opportunity to study differences between species that were formerly possible only using uni-algal cultures. The objective was to study differences in the photochemical efficiency of photosystem II (F v /F m ) between different species or different genera (when identification to the species level was not possible) and to investigate the effect of salinity on individual species. The difference in F v /F m between species was small, but often significant (ANOVA, p < 0.05), and was found to be a true characteristic of species, as opposed to being caused by stochastic processes affecting cells of all species more or less equally. High algal mortality is implied by a large decline of biomass from freshwater towards higher salinity, as well as a complete change in the phytoplankton composition. Nevertheless, cells with low F v /F m , which would indicate decreased health, were rare. F v /F m of 93% of the measured cells was between 0.5 and 0.8. There was no obvious relation between F v /F m and species abundance. In laboratory studies, green algae are often reported to have a higher F v /F m than diatoms, but we found no indication of this in the Scheldt estuary.