Abstract The organic matter (OM) of surface sediments (0–2 cm) from the Santos Basin was characterized by employing elemental (C, N, P), isotopic (δ 13C and δ 15N), biochemical (total carbohydrates, proteins, and lipids), and pigment (chlorophyll-a and phaeopigments) proxies, as part of the Santos Project – The Santos Basin Regional Environmental Characterization (PCR-BS) – coordinated by PETROBRAS. Samples were collected in a regional scale throughout the entire basin, including 11 isobaths from eight cross-margin transects ranging from 25 to 2,400 m water depths, and 12 additional samples obtained from the São Paulo Plateau. The aims of this study were two-fold, namely, to identify the major oceanographic and depositional processes driving OM transport and accumulation and to provide information on OM origin and availability to benthic secondary producers. The multiple employed proxies and the use of machine learning allowed to categorize distinct OM accumulation provinces displaying two major regional features: shallow sediments (< 50-m isobaths) are OM-poor (TOC < 2.0 mg g -1) but the available amount is readily hydrolysable, and mid-shelf deposits (75-m and 100-m isobaths) throughout the entire basin present muddy and OM-rich sediments (TOC ranging from 7.0 to 14.0 mg g-1) seemingly available to heterotrophs. Similarly, relatively OM-rich deposits are found at depths ranging from 700 to 1,300-m isobaths throughout the basin, but their composition and nutritional value (as suggested by biochemical indicators) are not related to mid-shelf deposits. The general overview of OM accumulation and composition provided herein validated the selected organic geochemical proxies for benthic ecology studies at the shelf and deep ocean sectors of the studied region.