Despite the popularity of stable isotope analysis (Carbon and Nitrogen), the drivers of species isotopic niches and their consequences on food web functioning remained poorly described, especially in estuaries. Here, we hypothesised that species niche characteristics are influenced by ecosystem hydro-morphological features and the functional structure of fish assemblages. The trophic niches of four fish species (Dicentrarchus labrax, Pomatoschistus minutus, Sprattus and Platichthys flesus) and overlap between them were compared during two consecutive autumns in eight estuarine ecosystems of the northeast Atlantic Ocean. Our findings demonstrated a relative steadiness of trophic positions of fish in estuaries, despite a high level of interspecific variability. The seabass generally occupied the higher trophic position, followed by the sand goby, while the flounder and sprat were at the lowest position in the food web. Species isotopic niches and overlap were mediated by some estuarine features, including estuary size and intertidal extent, but also by biotic interactions at the intra- and inter-specific levels, as reflected by the influence of fish abundance and functional diversity metrics. Our results support the statement that the intertidal area is a pivotal factor in regulating trophic interactions, by promoting niche partitioning and diversification of trophic resources between species. Moreover, niche size and dietary divergence of species decreased with increasing regularity of trophic strategies within assemblages, suggesting that species occupied more restricted trophic niches when they were less subjected to competition for the same food resources. These findings emphasize the importance of local conditions and the functional composition of assemblages in modulating the trophic resources consumed by fish in estuarine environments.