Viruses and small heterotrophic flagellates are known to exert an important control on bacterial populations. In parallel with the study of picoplankton dynamics (abundance and distribution) in surface waters (0–50 m) of Lakes Geneva, Bourget and Annecy, we used a dilution technique during different seasonal periods in order to assess flagellate- versus virus-induced mortality of heterotrophic bacteria, picocyanobacteria and small eukaryotic phytoplankton. Although it was not always possible to detect a significant viral effect (typically in winter), viral lysis and protozoan grazing could be responsible for up to 71% of the bacterial mortality (in summer). Viral impact, considered alone, never equalled or exceeded predation for heterotrophic bacteria, but could for picocyanobacteria, typically in autumn. In addition, during summer, complex interactions between grazing- and virus-induced mortality of bacteria (e.g. synergism versus antagonism) could be highlighted (for instance with bacterial lysis susceptible to enhance picocyanobacterial growth). The temporal variations observed for experimental viral parasitism and flagellate predation were consistent with the in situ dynamics and statistical relationships found between the targeted communities. This study thus provides new evidence on the critical role played by viruses and small flagellates in the functioning of freshwater microbial food webs and also that these mortality processes vary strongly throughout the seasons.