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The flux of organic matter (OM) across ecosystem boundaries can influence estuarine food web dynamics and productivity. However, this process is seldom investigated taking into account all the adjacent ecosystems (e.g. ocean, river, land) and different hydrological settings (i.e. river discharge). Therefore, we aimed to quantify the contribution of autochthonous and allochthonous OM to the lower food web along the estuarine salinity gradient, under different river discharge conditions. The carbon (C) and nitrogen (N) stable isotope ratios of pelagic (zooplankton) and benthic (Corbicula fluminea) primary consumers indicated that they rely on a mixture of autochthonous and allochthonous OM, including terrestrial-derived OM. Unexpectedly, the highest contribution of terrestrial-derived OM to the estuarine food web was observed during a low river discharge period (up to 70%), that succeeded a large winter flood, showing that extreme weather events may produce prolonged effects on estuarine food webs. The contribution of marine-derived OM was higher during low river discharge periods (up to 88%) and was restricted to the seaward end of the estuary. Concomitantly, the contribution of phytoplankton to primary consumers was the highest observed (up to 91%). Further, both pelagic and benthic consumers also relied on benthic C (i.e. sediment OM and microphytobenthos). This study demonstrates that primary consumers enhance connectivity between estuarine ecosystems by utilizing subsidies of terrestrial and marine origin, and also between estuarine habitats through reliance on pelagic and benthic OM.