© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd Understanding the ecosystem functioning in the dark portion of the ocean is a challenge that microbial ecologists are still facing. Due to the large volume, the global deep Ocean plays a central role in the regulation of climate, possibly buffering the rise of atmospheric carbon dioxide if processes of CO2 fixation compensate for respiration. We investigated the rates of several prokaryotic activities (dissolved and particulate primary production, heterotrophic carbon production and respiration) in meso- and bathypelagic waters of the Mediterranean Sea, covering all sub-basins. Chemosynthesis was the main process for C uptake. The rates of organic C (OC) excretion (or viral-induced cell lysis) inferred from the dissolved primary production measurements were noteworthy, being comparable to particulate primary production, and possibly contributing to the formation of non-sinking particulate organic matter. Inorganic C fixation rates were significantly higher than those reported for other deep-sea systems, probably as a consequence of the persistently higher temperature of dark Mediterranean waters or to phylogenetically diverse communities involved in the process. Primary production was negatively correlated with dissolved organic carbon concentration and showed an inverse pattern to heterotrophic carbon production, indicating a niche partitioning between heterotrophs and autotrophs. In sum, the deep Mediterranean Sea harbors active autotrophic communities able to fix inorganic carbon faster than the heterotrophic carbon production rates.