An interdisciplinary and multiagency study was completed to understand harmful algal bloom dynamics by describing the spatial and temporal patterns and drivers of phytoplankton community structure and cyanotoxin and cyanotoxin-gene concentrations in Lake Okeechobee and Okeechobee Waterways in South Florida. The Waterway includes the Caloosahatchee and St. Lucie Rivers which drain the lake westward to the Gulf of Mexico and eastward to the Atlantic Ocean, respectively. Algae and cyanobacteria play crucial roles in aquatic ecosystems, but under favorable environmental conditions certain taxa experience population growth booms resulting in harmful algal blooms. Cyanobacteria are considered the most common bloom-formers in freshwaters and can be particularly devastating due to the diversity and potency of the cyanotoxins they can produce. A dataset was compiled that contains phytoplankton community composition and abundance data for 21 sites sampled monthly from March 2019 through October 2021. The 21 sites include 17 sites in Lake Okeechobee and 4 sites along the upper and lower reaches of the Caloosahatchee and St. Lucie Rivers. The datafile provides algal identification, enumeration, biovolume, and functional group data at the genus level.