Subtropical estuaries worldwide face increased pressure on their ecosystem health and services due to increasing human population growth and associated land use/land cover changes, expansion of ports, and climate change. We investigated freshwater inflows (river discharge) and the physico-chemical characteristics of Galveston Bay (Texas, USA) as mechanisms driving variability in phytoplankton biomass and community composition between February 2008 and December 2009. Results of multivariate analyses (hierarchical cluster analysis, PERMANOVA, Mantel test, and nMDS ordination coupled to environmental vector fitting) revealed that temporal and spatial differences in phytoplankton community structure correlate to differences in hydrographic and water quality parameters. Spatially, phytoplankton biomass and community composition responded to nutrient loading from the San Jacinto River in the northwest region of the bay (consistent with nutrient limitation) while hydraulic displacement (and perhaps other processes) resulted in overall lower biomass in the Trinity River delta (northeast region). The influence of inflows on phytoplankton diminished along a north to south gradient in the bay. Temporally, temperature and variables associated with freshwater inflow (discharge volume, salinity, inorganic nitrogen and phosphorus concentrations) were major influences on phytoplankton dynamics. Dissolved inorganic nitrogen: phosphorus (DIN:DIP) ratios suggest that phytoplankton communities will be predominately nitrogen limited. Diatoms dominated during periods of moderate to high freshwater inflows in winter/spring and were more abundant in the upper bay while cyanobacteria dominated during summer/fall when inflow was low. Given the differential influences of freshwater inflow on the phytoplankton communities of Galveston Bay, alterations upstream (magnitude, timing, frequency) will likely have a profound effect on downstream ecological processes and corresponding ecosystem services.