Laguna de Rocha belongs to a series of shallow coastal lagoons located along South America. It is periodically connected to the sea through a sand bar, exhibiting a hydrological cycle where physicochemical and biological gradients are rapidly established and destroyed. Its most frequent state is the separation of a Northern zone with low salinity, high turbidity and nutrient load, and extensive macrophyte growth, and a Southern zone with higher salinity and light penetration, and low nutrient content and macrophyte biomass. This zonation is reflected in microbial assemblages with contrasting abundance, activity, and community composition. The physicochemical conditions exerted a strong influence on community composition, and transplanted assemblages rapidly transformed to resembling the community of the recipient environment. Moreover, the major bacterial groups responded differently to their passage between the zones, being either stimulated or inhibited by the environmental changes, and exhibiting contrasting sensitivities to gradients. Addition of allochthonous carbon sources induced pronounced shifts in the bacterial communities, which in turn affected the microbial trophic web by stimulating heterotrophic flagellates and virus production. By contrast, addition of organic and inorganic nutrient sources (P or N) did not have significant effects. Altogether, our results suggest that (i) the planktonic microbial assemblage of this lagoon is predominantly carbon-limited, (ii) different bacterial groups cope differently with this constraint, and (iii) the hydrological cycle of the lagoon plays a key role for the alleviation or aggravation of bacterial carbon limitation. Based on these findings we propose a model of how hydrology affects the composition of bacterioplankton and of carbon processing in Laguna de Rocha. This might serve as a starting hypothesis for further studies about the microbial ecology of this lagoon, and of comparable transitional systems.