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Planktonic microbial communities mediate many vital biogeochemical processes in wetland ecosystems, yet compared to other aquatic ecosystems, like oceans, lakes, rivers or estuaries, they remain relatively underexplored. Our study site, the Florida Everglades (USA)—a vast iconic wetland consisting of a slow-moving system of shallow rivers connecting freshwater marshes with coastal mangrove forests and seagrass meadows—is a highly threatened model ecosystem for studying salinity and nutrient gradients, as well as the effects of sea level rise and saltwater intrusion. This study provides the first high-resolution phylogenetic profiles of planktonic bacterial and eukaryotic microbial communities (using 16S and 18S rRNA gene amplicons) together with nutrient concentrations and environmental parameters at 14 sites along two transects covering two distinctly different drainages: the peat-based Shark River Slough (SRS) and marl-based Taylor Slough/Panhandle (TS/Ph). Both bacterial as well as eukaryotic community structures varied significantly along the salinity gradient. Although freshwater communities were relatively similar in both transects, bacterioplankton community composition at the ecotone (where freshwater and marine water mix) differed significantly. The most abundant taxa in the freshwater marshes include heterotrophic Polynucleobacter sp. and potentially phagotrophic cryptomonads of the genus Chilomonas, both of which could be key players in the transfer of detritus-based biomass to higher trophic levels.