This study aims to evaluate the seasonal and interannual variability in the importance of the microbial food web in Lake Tanganyika. Phytoplankton, bacteria and protozoa (heterotrophic nanoflagellates and ciliates) were monitored at two contrasting pelagic sites (Kigoma and Mpulungu) during 3 consecutive years. In addition, spatial variation was studied along 3 north-south transects during contrasting seasons. The study period covered a wide range of limnological conditions, with mixing depth ranging from 13 to >100 m and euphotic depth from 14 to 65 m. The consistently high bacterial biomass (up to 62 μg C l−1 ) and the high contribution of small phytoplankton (< 5 μm) to the total phytoplankton biomass (on average 50 % in Kigoma and 84 % in Mpulungu) point to an important role of the microbial food web in the lake throughout the year. Total phytoplankton biomass increased during periods of low water column stability, with an increased biomass of small eukaryotic phytoplankton (2 to 5 μm) at both stations, together with autotrophic prokaryotic picoplankton at the southern station Mpulungu and diatoms at the northern station Kigoma. Heterotrophic bacteria, heterotrophic nanoflagellates (0.06 to 11.01 μg C l−1 ) and ciliates (up to 8.16 μg C l−1 ) did not show this seasonality. The main seasonal and spatial variability in the importance of the microbial food web seems therefore primarily linked to the contribution of small phytoplankton, which may be better adapted to lower average light intensities and higher N:P ratios during periods of deep mixing.