Sediments from Guanabara Bay, sampled as short-cores (50–60 cm) from eight stations, were analysed for the elemental (C and N), isotopic (δ13C and δ15N) and molecular composition (steroids) of the organic matter. The objective of this study was to examine whether there are changes in the sources of sedimentary organic matter pool due to increasing N, P and particulate loads to the bay over the last 100 years. On average, we found a 10-fold increase in the flux of organic matter to the sediments with a maximum of 41.7 mol C m−2 year−1. C/N and δ13C time–space distribution and mass balance calculations indicate that the organic material results from a mixture of marine, terrestrial and estuarine sources, with a growing predominance of autochthonous inputs in recent years. Dinosterol was predominant in most samples (>50% of all measured sterols). Concentrations of dinosterol increased significantly in the last two decades and reached values as high as 150 μg g−1 dry sediment. Net increases in concentration were estimated using the first-order decay rate constants derived from experimental data. There are strong indications that an increase in carbon storage occurred in response to growing eutrophic conditions. In spite of the high respiration rates occurring in the water column, fast sedimentation rates result in the transfer of a significant carbon fraction to the anoxic sediments. Further decomposition proceeds only in the first 10 cm of sediment as indicated by the decay rates.