We examined the carbon sources used by bacteria and mesozooplankton in the Scheldt River and estuary (Belgium, The Netherlands) using a combined stable isotope and fatty acid composition approach. Water samples were collected monthly at six stations during 1 yr and analyzed for 13C of dissolved inorganic carbon, dissolved organic carbon (DOC), and particulate organic carbon (POC). Mesozooplankton was determined up to family, genus, or species level and analyzed for 13C and fatty acid content. Suspended particles were analyzed for phospholipid fatty acids and their 13C contents to estimate isotope ratios of phytoplankton groups and heterotrophic bacteria. The carbon isotope signatures of DOC, POC, and bacterial biomass were similar and significantly enriched relative to those of diatoms and green algae, pointing to allochthonous subsidies as an important carbon source for bacteria. The contribution of algae to zooplankton diets as estimated from isotope ratios and fatty acid profiles averaged 41% and 75% respectively, and did not differ significantly among stations, taxa, or age categories. Mesozooplankton relies primarily on grazing on phytoplankton and direct consumption of particulate organic matter. Mesozooplankton appears to receive little of its carbon from DOC via bacteria.