The Paraíba do Sul River (PSR), in the SE Brazilian region, has been severely altered by multiple human activities. To evaluate the influence of the PSR on the organic matter (OM) pool in sediments of the shelf area off the estuary, bulk stable isotopic composition (δ13C) and lipid biomarkers were studied in 64 surface samples. δ13C in the OM ranged from -26.9‰ to -20.3‰ and from -27.1‰ to -21.1‰ in the rainy and dry seasons, respectively. A principal component analysis of individual biomarkers suggests preferential accumulation of autochthonous and labile OM in the shallow shelf environment (< 30 m depth), mainly in the rainy season. This is ascribed to enhanced primary production over the shelf supported by nutrients delivered during periods of high river flow. Regarding terrestrial OM, the δ13C and lipid biomarkers appear to represent distinct pools of OM, respectively those associated with coarse debris and fine particles. Such differences might result from distinct degradation rates and transport mechanisms along the river-estuary-coastal sea continuum. Our data reveal how complex and dynamic the nature and distribution of OM can be in sandy and C-poor sediments adjacent to a medium-sized river as a consequence of anthropogenic and natural factors.