The microbial plankton community is an integral part of the pelagic ecosystem. It hosts essential functional groups that play a vital role in organic carbon production, release, uptake, and degradation within open-ocean ecosystems. Given its significance, carbon biomass estimates are urgently needed, especially in oligotrophic regions, to provide and enhance our knowledge of biogenic carbon pools. They also aid in validating biogeochemical models that characterize the functioning of these extensive marine ecosystems within the global carbon cycle. This study addresses the temporal variability of microbial community biomass in two oceanic zones: the west-central (Perdido) and southern (Coatzacoalcos) areas of the Gulf of Mexico. During three seasonally contrasting periods (nortes, rainy, and dry seasons), seawater samples were collected from the euphotic zone in both regions to estimate the carbon biomass of different pico- (<2–3 µm), nano-, and microplankton groups (>3–200 µm). Carbon biomass assessments for the microbial groups were based on their abundance and carbon conversion factors. Overall, we found a significant contribution of pico-prokaryotic components (heterotrophic bacteria, Prochloroccocus, and Synechoccocus) to the total microbial carbon stock of the euphotic zone (84–89 % global estimates). The finding suggests these microorganisms are key functional groups that drive carbon production and fate in the Gulf of Mexico ecosystem. Pico-cyanobacteria, especially Prochloroccocus, were the dominant primary producers (68–82 % total autotrophic carbon), mainly in the upper layer of the oligotrophic euphotic zone. This vertical pattern implies that the deep chlorophyll-a maximum (DCM) depth level was unrelated to a net increase in phytoplankton biomass in the three study periods. The distribution of microbial carbon biomass exhibited striking differences associated with winter mixing (the nortes season), high river discharge accompanied by cross-shelf transport (the rainy season), and the dynamics of mesoscale structures. Ecological aspects, such as the habitat preference of the organisms and the seasonal complementary development of mixotrophic and heterotrophic grazers and their prey, were also essential drivers in regulating the microbial carbon pool of both oceanic regions. The microbial carbon assessments conducted in this study contribute to identifying and quantifying key planktonic functional groups involved in the biogeochemical carbon cycle in the Gulf of Mexico open-ocean ecosystem.