Most of the ocean is deep with the majority of its volume (> 80%) lying under a depth greater than 1000 m. Deep-ocean substrates input is mainly supplied as organic matter (in particulate and/or dissolved forms) by physical and biological processes. Bioavailable dissolved organic carbon (DOC) is mainly consumed in surface water by prokaryotes, while most of DOC in the deep ocean is recalcitrant. Deep-sea prokaryotes are known to be adapted to degrade complex substrates. In this study, we investigate the utilization of HMW-DOC on the short temporal scale (10–15 days) by deep-sea prokaryotes maintained at in situ high-pressure conditions. Deep-sea prokaryotic natural assemblages were collected in the Mediterranean Sea in two contrasting hydrological conditions (water column stratification and deep-water formation period conditions). The experimental results were coupled with a cell-quota model, in order to quantify the kinetics of HMW-DOC degradation and its impact on the prokaryotic assemblages under these two contrasting hydrological conditions. The results show that under stratified water conditions autochthonous deep prokaryotic assemblages are able to degrade up to 46.6% of DOC on the timescales of the incubation, when maintained under in situ sampling high-pressure conditions. By contrast, during deep-water convection period condition, DOC is weakly degraded on the timescales of the incubation under in situ high-pressure conditions. This study shows that the remineralization rates of DOC are controlled by the prokaryotic communities, which are further driven by the hydrological conditions of the water column.