Estuarine fish assemblages are subject to a great environmental variability that largely depends on both upstream fluvial and downstream marine influences. From this ecohydrological view, our study introduces a macroecological approach aiming to identify the main environmental factors that structure fish assemblages among European tidal estuaries. The present paper focuses on the influence of large scale environmental gradients on estuarine fish species richness. The environment of 135 North-eastern Atlantic estuaries from Portugal to Scotland was characterized by various descriptors especially related to hydromorphology. Major environmental trends among estuaries were underlined using multivariate techniques and cluster analyses applied to abiotic data. In particular, an integrative system size covariate was derived from a principal component analysis. Factors explaining patterns of species richness at different scales from local habitat to regional features were highlighted. Based on generalised linear models, the estuarine system size, and more particularly the entrance width, and also the continental shelf width were identified as the best explanatory variables of estuarine fish species richness at a large scale. Our approach provides a standardized method to estimate the relationship between fish assemblages and environmental factors. This constitutes a first step in assessing estuarine ecological status and studying the effects of additional factors such as anthropogenic disturbances.