This dataset consists of a variety of hydrographic, biological, biogeochemical and meteorological data. Underway automatic sampling followed by flow cytometry was used for assessing mesoscale spatial variability of microorganisms. Coarse-scale (CTD) and fine-scale (PumpCast profiler) vertical distribution of microorganisms was determined, with dominant microbial prokaryotic and eukaryotic groups quantified by flow cytometry. Samples were collected and processed for subsequent molecular identification of flow cytometrically sorted dominant microbes. Abundance of larger microplankton organisms was assessed. Microbial respiration in the twilight zone was studied. Group-specific uptake of bicarbonate, phosphate and different nitrogen compounds was determined and group-specific production and grazing assessed using flow sorting. The ambient turnover rates of phosphate, organic phosphorus and labile dissolved organic matter, e.g. amino acids, was bioassayed. In order to put the microbial community data into context, physical and chemical parameters were measured. A ship mounted Acoustic Doppler Current Profiler (ADCP) was used to collect information about physical, mesoscale spatial context and, when combined with turbulence profiler data, SeaSoar profiler data and data collected using intensive Conductivity, Temperature and Depth (CTD) sampling for dissolved inorganic nutrients, may estimate mesoscale nutrient fluxes into the photic zone. PELAGRA neutral buoyancy sediment traps were used to estimate biogenic sedimentation. Samples were collected for particulate calcite, opal, and Particulate Organic Matter (POM) measurements, together with the isotopic composition of Particulate Organic Nitrogen (PON). Data collection was in the North Atlantic subtropical gyre between 9th August and 15th September 2011 during RRS Discovery cruise D369. The data were collected as part of the United Kingdom (UK) Natural Environment Research Council (NERC) Oceans 2025 programme (Theme 2). D369 was the last of three National Oceanography Centre 'process study' research cruises to be run by the Ocean Biogeochemistry and Ecosystems research group under the NERC Ocean 2025 research programme. The Principal Scientist was Mikhail V Zubkov of National Oceanography Centre, Southampton. Data from this cruise are held at the British Oceanographic Data Centre.