•The Danube water quality was predicted using macrophyte binary data.•The developed model showed good predictive performances in the main river channel.•The sensitive analysis selected 28 macrophytes as significant water quality indicators. Ecological assessment of large rivers such as the Danube is a challenging task. Eutrophication was reported as one of the main drivers that structure aquatic communities in the Danube basin. Due to their sedentary nature, relatively slow growth/ long life spans, and engineering role in aquatic ecosystems, macrophytes are widely used in the detection of nutrient enrichment. In this study, macrophyte presence-absence data within the 3 km long reaches obtained from the Joint Danube Survey (JDS3) were used to predict the water quality of the Danube river and its main tributaries. For each water quality variable (dissolved oxygen, nitrate-nitrogen, and orthophosphates), a multi-layer feed-forward artificial neural network model (ANN) was constructed using the macrophytes as explanatory variables. Despite the limited number of samples (123) along the wide trophic gradient of the Danube, the model showed good predictive performances for the main river channel. The highest discrepancy between observed and predicted water quality was obtained for the samples collected in the tributaries or downstream from the tributaries' mouth, where the model predicted better trophic conditions compared to measured ones. From 64 analysed macrophyte species, 28 were selected by sensitivity analysis as key water quality indicators (KIS) for at least one environmental variable. KIS mainly belonged to the eutrophic tolerant submerged or emerged species with broad ecological amplitude, which reflects the significance of the developed model for use on rivers subjected to nutrient pollution. However, the use of the developed predictive model is restricted to the river sections having a water velocity suitable for macrophytes growth. The developed ANN architecture represents the modelling approach which could be applied to other lotic systems and biological quality elements.