We investigated the distribution and dynamics of viruses, prokaryotes and small eukaryotic phytoplankton in Sidi Salem freshwater reservoir (Northern Tunisia). Samples were collected from the deepest station at different depths throughout the water column for 2 years (February 2009 to January 2011). The reservoir was characterized by seasonal alternations of thermal stratification and homothermy. Among the different microbial communities counted using flow cytometry (FCM), picocyanobacteria constituted an important autotrophic component since they were always present and their highest concentration reached 3.02 and 2.65 x 10(5) cells.mL(-1) in March 2009 and June 2010, respectively. The heterotrophic prokaryotic communities (represented mainly by bacteria) were characterized by a clear separation between two subgroups referred to as high-DNA and low-DNA content populations, and the highest concentrations of heterotrophic bacteria (i.e., 3.8 x 10(7) cells.mL(-1)) were recorded in spring 2009. Several viral groups referred to as virus-like particles (VLP) groups 1, 2 and 3 could also be discriminated using FCM. VLP1 and VLP2 displayed a significant correlation with the heterotrophic bacteria (r = 0.80 and 0.78, P < 0.001) but seem to be independent from picocyanobacteria and/or chlorophyll a, suggesting these VLPs were mainly bacteriophages. At last, the virus to prokaryotic ratio could be high, especially in summer (mean = 22, max = 487), suggesting a strong coupling between bacteria and viruses, at least at certain periods of the year.