Phytoplankton and bacterioplankton represent the base of the aquatic food webs. The changes in the structure of their communities at different levels, from taxonomic and functional composition to single cells traits, and the degree of coupling among them has strong impact on transfer of energy to higher trophic levels and finally on the fate of organic matter. Changes in the structural pattern of the microbial community at different levels have been studied in temperate estuaries, but are poorly characterized in their tropical counterparts. Here, we show for the first time changes in single-cell characteristics and abundance and of key components of the planktonic microbial food web (picocyanobacteria, photoautotrophic pico- and nanoeukaryotes and heterotrophic bacteria) during the rainy and dry seasons along the estuarine gradient of the inner Gulf of Nicoya, one of the most productive estuaries in the world. The different responses of the microbial functional assemblages changed the structure of the pelagic microbial community, which likely affects higher trophic levels and the ecosystem functioning. Moreover, we relate the changes in the community structure to differences in environmental conditions along the estuary, during both seasons. Multivariate ordinations based on the microbial assemblages revealed a stronger covariation among them and with environmental variables during the dry season. The relative decrease in the inputs of allochthonous organic carbon and the increase in solar radiation during the dry season likely induced a higher degree of coupling between the low size phyto- and bacterioplankton assemblages along the tropical estuarine gradient due to increased local production.