The iAtlantic Project has established an international collaborative strategy to improve mapping and characterization of deep and open ocean ecosystems in understudied regions of the Atlantic and evaluate their health. In December 2022, the first iAtlantic expedition in the South Atlantic set off to map and explore seafloor ecosystems in the Santos Basin slope (200-1,000 m depths) in collaboration with the Petrobras ‘Santos Basin - Regional Characterization Project.’ The 17-day ‘iAtlantic_BR10-Petrobras’ cruise was conducted on board the research vessel NPqHOc Vital de Oliveira (Brazilian Navy) and performed (a) water column structure characterization, (b) seafloor morphology mapping, (c) description of benthic habitats and communities by seafloor imagery and biological/ geological sampling, and (d) ex-situ experiments to assess the functioning of sedimentary ecosystems and their responses to climate-related environmental changes. This study describes the rationale behind the iAtlantic_BR10-Petrobras cruise science plan, reports its sampling strategy and methods, and summarizes its collected data and preliminary results.