Biogeochemical markers in combination with bacterial community composition were studied at two contrasting stations at the Río Negro (RN) estuary to assess the outwelling hypothesis in the Argentinian Patagonia. Inorganic nutrients and dissolved organic matter were exported clearly during the last hours of the ebb at the station Wetland. Moreover, a considerable outwelling of polyunsaturated fatty acids (PUFA), particulates and microalgae was inferred by this combined approach. The exported 22:6(n-3) and 20:5(n-3) contributed very likely to sustain higher trophic levels in the coasts of the Southwest Atlantic. The stable isotopes did not evidence clearly the outwelling; nevertheless, the combination of δ13C with fatty acid bacterial markers indicated organic matter degradation in the sediments. The dominance of Desulfobacterales and Desulfuromonadales suggested sulphate reduction in the sediments, a key mechanism for nutrient outwelling in salt marshes. Marivivens and other Rhodobacterales (Alphaproteobacteria) in the suspended particulate matter were clear indicators of the nutrient outwelling. The colonization of particles according to the island biogeography theory was a good hypothesis to explain the lower bacterial biodiversity at the wetland. The copiotrophic conditions of the RN estuary and particularly at the wetland were deduced also by the dynamic of some Actinobacteria, Bacteroidia and Gammaproteobacteria. This high-resolution snapshot combining isotopic, lipid and bacterial markers offers key pioneer insights into biogeochemical and ecological processes of the RN estuary.