This project aimed to conduct an initial microbial community survey to assess prokaryotic diversity and potential seasonal patterns in surface waters of the Caloosahatchee River and Estuary, a dynamic freshwater-estuarine system in southwest Florida. Understanding microbial assemblages in these environments contributes to long-term ecological monitoring, nutrient cycling studies, and water quality assessments. Surface water samples were collected from three long-term monitoring sites in the Caloosahatchee River and Estuary, Fort Myers, Florida. Water samples were collected by the Lee County Environmental Laboratory. Genomic DNA was extracted from filtered samples and submitted to the University of Hawaii Advanced Studies in Genomics, Proteomics, and Bioinformatics (ASGPB) facility for 16S rRNA amplicon library preparation and sequencing using the Illumina MiSeq platform.