Flow-regulated discharges of water from control structures into estuaries result in hydrologic and water chemistry conditions that impact spatial and temporal variability in the structure and biomass of phytoplankton communities, including the potential for harmful algal blooms (HABs). The relationships between regulated Caloosahatchee River (i.e., C-43 Canal) discharges and phytoplankton communities in the Caloosahatchee Estuary and adjacent nearshore regions on the southwest coast of Florida were investigated during two study periods, 2009–2010 and 2018–2019. During periods of low to moderate discharge rates, when mesohaline conditions predominated in the estuary, and water residence times were comparatively long, major blooms of the HAB dinoflagellate species Akashiwo sanguinea were observed in the estuary. Periods of high discharge were characterized by comparatively low phytoplankton biomass in the estuary and greater influence of a wide range of freshwater taxa in the upper reaches. By contrast, intense blooms of the toxic dinoflagellate Karenia brevis in the nearshore region outside of the estuary were observed during high discharge periods in 2018–2019. The latter events were significantly associated with elevated levels of nitrogen in the estuary compared to lower average concentrations in the 2009–2010 study period. The relationships observed in this study provide insights into the importance of managing regulated discharge regimes to minimize adverse impacts of HABs on the health of the estuary and related coastal environments.