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In this study, we carried out dilution experiments at the surface and in the mesopelagic and bathypelagic layers at 15 sites in the Mediterranean Sea that covered a wide range of trophic conditions. The main aim was to test the hypothesis that prokaryotes, and particularly heterotrophic prokaryotes, are pivotal in sustaining both nanoplankton and microzooplankton energy requirements at all of the considered trophic states. These data highlight that bacterivory is the major pathway of organic carbon transfer in the oligotrophic and meso-eutrophic environments. The microzooplankton mostly feed on prokaryotes, directly or indirectly (through nanoplankton exploitation), rather than on microalgae. Under eutrophic conditions, herbivory is the main trophic pathway; however, the heterotrophic prokaryotes always represent an important source of carbon. The lowest food-web efficiency (i.e., ratio between productivity of the highest trophic level and productivity of the lower trophic levels) was determined for the eutrophic status due to possible grazer satiation, which translates into an excess of autotrophic biomass available for export or transfer to higher trophic levels. The food-web efficiency is higher under mesoeutrophic and oligotrophic conditions, where the main pathway is bacterivory. In the mesopelagic and bathypelagic layers, only nanoplankton predation on heterotrophic prokaryotes was investigated. The food-web efficiency in these layers was relatively high and nanoplankton appear to efficiently exploit the available biomass of heterotrophic prokaryotes.