The estuarine phytoplankton communities are known to respond rapidly to environmental changes, being considered an important water quality indicator; thus, it is crucial to fully understand its natural variability. The objective of the present study was to assess the spatial variability of both physicochemical variables and the phytoplankton community, to understand how such variability is influenced by seasonality and to evaluate how the anthropogenic sources affect such patterns. The Tagus estuary was used as a case study, since it is one of Europe’s largest estuaries, with high spatial and seasonal variations and a high level of human pressure associated with large urban and industrial areas. To achieve this goal, environmental parameters, nutrients concentration, bivalve biomass (filter feeders) and phytoplankton pigments were quantified in a single summer sampling campaign with high spatial resolution and in monthly campaigns in eight sampling stations through the Tagus estuary, in one year. In general, suspended particulate matter and nutrients decreased from the upper part of the estuary to the estuary mouth; however, relevant local inputs were also observed in more downstream locations, near outfalls of wastewater treatment plants (WWTP). The chlorophyll-a concentrations were higher over the southern intertidal mudflats, probably due to resuspension of microphytobenthos, associated with higher nutrient concentrations. Through a grazing indicator, it was observed that grazers are important drivers of variability of the phytoplankton community composition. All water bodies achieved “good” and “high” water quality classifications for both physicochemical and biological indicators, with the worst results reported for the water bodies located at the upper estuary. Therefore, this estuary presents a decreasing trend of nutrients and chlorophyll-a in the upstream–downstream direction, except for the estuary channels and the outfall in the northern margin, which lead to an increase in nutrient concentrations. However, these increases did not affect the water quality of the three analyzed water bodies, presenting at least good ecological status, considering the nutrient and chlorophyll-a indicators.