The delta of the Parnaíba River has a set of highly dynamic, productive ecosystems located in the Amazon–semiarid interface environment on the coast of Brazil. Phytoplankton is a key element in the evaluation of aquatic environments, as it is extremely sensitive to the broad spatial and seasonal variability of masses of freshwater and the physicochemical conditions of the water. To evaluate spatial–seasonal variation in the hydrological characteristics and the phytoplankton community in relation to the saline gradient, three sampling campaigns were performed at eight points considering seasonal periods (dry, rainy and transition) and spatial distribution (Nearshore Turbid Zone [NTZ], Mixing Zone [MZ] and Tidal River Zone [TRZ]) during neap tides. Variability in the environmental characteristics and phytoplankton was tested using permutational multivariate analysis of variance, dissimilarities and correlations. The data revealed a strong spatial–seasonal influence throughout the entire length of the estuary, with a clear spatial gradient related to salinity. Thalassiosira subtilis was the bioindicator for the NTZ, Cyclotella meneghiniana for the MZ and Oocystis parva for the TRZ. Concentrations of chlorophyll a, suspended particulate matter and nitrite were the main variables affected by the two factors combined (seasonality and spatial distribution). Moreover, total dissolved solids, nitrate and nitrite were the variables that promoted favorable ecological conditions for the development of the phytoplankton community associated with seasonality, especially the growth of Cyclotella meneghiniana, which proved to be better adapted to these conditions and was considered a key species in the area studied.