The present work aimed at studying the origin of particulate organic matter in Guanabara Bay and in some rivers of the Guanabara basin by using elemental composition, isotopic ratios (δ13C and δ15N) and molecular markers (sterols) in samples collected in two periods (winter and summer). Elemental and isotopic compositions were determined by dry combustion and mass spectrometry, respectively, while sterols were investigated by GC–FID and GC–MS. Higher sterol concentrations were present in the north-western part of the bay in winter (5.10–23.5 μg L–1). The high abundance of algal sterols (26–57% of total sterols), the elemental composition (C/N=6–8) and the isotopic signatures (δ13C=−21.3‰ to −15.1‰ and δ15N=+7.3‰ to +11.1‰) suggested the predominance of autochthonous organic matter, as expected for an eutrophic bay, although seasonal variation in phytoplankton activity was observed. Coprostanol concentration (fecal sterol) was at least one order of magnitude higher in the particulate material from fluvial samples (4.65–55.98 μg L–1) than in the bay waters (<0.33 μg L–1). This could be ascribed to a combination of factors including efficient particle removal to sediments in the estuarine transition zone, dilution with bay water and bacterial degradation during particle transport in the water column.