This dataset contains the results of the characterisation of the prokaryotic community by flow cytometry and tritiated leucine incorporation from the MAFIA cruise (Migrants and Active Flux In the Atlantic ocean). Samples were collected in the tropical and subtropical Atlantic during the MAFIA cruise (April 2015) on board the BIO Hespérides. Seawater samples were collected at 13 stations (from the Brazilian coast to the Canary Islands), from the surface down to 3500 m, using a General Oceanics oceanographic rosette equipped with 24 l PVC Niskin bottles. Abundance and cell characteristics (high nucleic acid content fraction, cell volume, viability) were based on measurements performed with a FACSCalibur (Becton-Dickinson) flow cytometer. Leucine incorporation rates were estimated with tritiated leucine (Kirchman et al. 1985) using centrifugation and filtration methods (Smith and Azam 1992). Integrated or average values of variables were estimated for the epipelagic (0-200 m), mesopelagic (200-1000 m) and bathypelagic (1000-3000 m) layers. The aim of this dataset was to estimate the influence of surface productivity on the standing stock, characteristics and activity (as leucine incorporation) of prokaryotes across the water column.