The extent to which water mass mixing contributes to the biological activity of the dark ocean is essentially unknown. Using a multiparameter water mass analysis, we examined the impact of water mass mixing on the nutrient distribution and microbial activity of the Northeast Atlantic Deep Water (NEADW) along an 8000 km long transect extending from 62°N to 5°S. Mixing of four water types (WT) and basin scale mineralization from the site where the WT where defined to the study area explained up to 95% of the variability in the distribution of inorganic nutrients and apparent oxygen utilization. Mixing-corrected average O2:N:P mineralization ratios of 127(±11):13.0(±0.7):1 in the core of the NEADW suggested preferential utilization of phosphorus compounds while dissolved organic carbon mineralization contributed a maximum of 20% to the oxygen demand of the NEADW. In conjunction with the calculated average mineralization ratios, our results indicate a major contribution of particulate organic matter to the biological activity in the NEADW. The variability in prokaryotic abundance, high nucleic acid containing cells, and prokaryotic heterotrophic production in the NEADW was explained by large scale (64–79%) and local mineralization processes (21–36%), consistent with the idea that deep-water prokaryotic communities are controlled by substrate supply. Overall, our results suggest a major impact of mixing on the distribution of inorganic nutrients and a weaker influence on the dissolved organic matter pool supporting prokaryotic activity in the NEADW.