To investigate the hypothesis of top-down control by viruses and heterotrophic nanoflagellates on bacterial-mediated carbon fluxes in freshwater systems, a year-long study (2023–2024) was conducted in the pelagic zone of Lake Saint-Gervais (France). The variability in BGE (9.9% to 45.5%) was attributed to the decoupling of production and respiration, providing bacterioplankton communities with a competitive advantage in adapting to fluctuating environmental disturbances in freshwater systems. The high nucleic acid (HNA) bacterial community, the active fraction, contributed the most to bacterial production and was linked to BGE estimates. Weak bottom-up controls (nutrient concentrations and stoichiometry) on BGE suggested a stronger role for mortality forces. Among viral subgroups (VLP1–VLP4) identified via flow cytometry, the dominant low-fluorescence DNA VLP1 subgroup (range = 0.7 to 3.1 × 108 VLP mL−1) accounting for the majority of viral production was closely linked to the HNA population. Both top-down forces exerted antagonistic effects on BGE at the community level. The preferential lysis and grazing of the susceptible HNA population, which stimulated bacterial community respiration more than production in the non-target population, resulted in reduced BGE. These results underscore the key role of top-down processes in shaping carbon flux through bacterioplankton in this freshwater system.