The abundance and biomass of heterotrophic nanoflagellates were examined in continental and deep-sea sediments of the Cretan Sea (Eastern Mediterranean); at depths of 40 to 1540 m. Nanoflagellate distribution was compared to the composition of sedimentary organic matter and bacterial standing stocks to investigate trophic interactions and factors potentially affecting distribution. Quantitative estimates were obtained using different samplers for testing whether the box corer is as effective as the multiplecorer for bacterial and protozoan population estimates. The sediments of the deep Cretan Sea appeared extremely deficient in organic nutrients, and were composed mostly (more than 90%) of detritus. Labile organic compounds (such as lipids, proteins, and soluble carbohydrates) were present at extremely low concentrations, decreasing with water depth. Refractory and structural carbohydrates were the dominant biochemical class. The decrease in food quality with depth was associated with a strong decline of the RNA:DNA ratio. Benthic bacteria were constrained by food availability, and reacted to different organic matter inputs (especially total carbohydrates) at different depths. Large size bacteria were significantly correlated with the amounts of proteins and chloroplastic pigments. Heterotrophic nanoflagellate distribution in the continental shelf and deep-sea sediments of the Cretan Sea was controlled by available food sources (i.e., labile organic compounds and bacteria). Flagellate density was significantly correlated with the concentration of food indicators (chlorophyll a, soluble carbohydrates, and lipids), and to bacterial number and biomass. Despite the oligotrophy of the system, flagellate densities were high (40–119 × 103 g−1) and dominated by small cells (3 to 6 μm in length). These results, coupled with the high nanoflagellate to bacterial biomass ratio (up to 0.27 at 40 m depth), suggest that benthic nanoflagellates may contribute significantly to the direct transfer of detrital carbon and bacterial biomass to the metazoan component of the food web in the Cretan Sea.