Dark carbon fixation (DCF) is considered an important energy source in aquatic environments, although it has been neglected for a long time. DCF is known to be relevant in ecosystems associated with redoxclines, shallow-water sulfide-rich habitats, deep-sea vents, cold seeps, and even in coastal waters associated with upwelling events. The aim of this study was to evaluate the relative importance of DCF in relation to heterotrophic bacterial production (BP), as well as how these rates affect each other, and how they are influenced by the environmental factors. This study was conducted monthly during 2 years in a tropical eutrophic bay (Guanabara Bay), where two stations were sampled and compared. DCF and BP were measured by C-14-bicarbonate and H-3-leucine incorporation, respectively, and incubations in the dark. Our results showed that DCF is not a quantitatively relevant process in this estuarine system, when compared to heterotrophic BP, and possibly occurred via anaplerotic reactions. Relatively higher DCF rates were associated with less oxygenated waters at the more polluted station and during the wet summer-spring, when the water column is more stratified. BP rates presented clear spatial patterns, according to pollution and depth gradients, with higher rates in more polluted areas, and also at surface waters. The hydrodynamics combined with other environmental conditions (precipitation, temperature, dissolved organic carbon, and nutrients) may control the distribution of DCF and BP over space and time. The allochthonous inputs of organic matter are more important than DCF-derived organic carbon to bacterioplankton in this polluted and eutrophic bay, where the heterotrophic metabolism prevails.