The main aim of this study was to evaluate the temporal changes in the phytoplankton community of the Tagus Estuary and to identify the stressors involved. Environmental and phytoplankton data were gathered from several studies conducted in the 1960s, 1980s and from 1999 to 2010 (2000s). Phytoplankton data included information on the community composition through microscopy. No significant change was found between temperature and nitrogen values in the three periods. Chlorophyll a concentrations varied throughout the years, and the lowest concentrations were observed after 2004. Significant differences were also found between phytoplankton cell abundances, lower in the 1980s compared to the ones recorded in the 2000s. In the 1980s, diatoms were the most abundant group in the majority of samples and were found to be associated with nitrogen concentrations (canonical correspondence analysis), which was not observed in the 2000s. In the period 2006–2007, the importance of diatoms decreased and smaller cells became more abundant (e.g. cryptophytes, euglenophytes, prasinophytes). The ratio cryptophyta/bacillariophyta seems to yield an increase from <1 in 1980s to >1 in 2006–2007. Mesodinium rubrum and Dinophysis produce recurrent toxic blooms in the adjacent coastal area. We can speculate that the estuary can be a cryptophyte producer to sustain the cryptophytes-M. rubrum-Dinophysis trophic relationship. A top-down hypothesis (shellfish grazing) is considered to explain the change in the phytoplankton community. A quantitative tool, the Phytoplankton Community Index (PCI), yielded a significant deviation of the community, from the 1980s to the 2000s, suggesting a shift toward the dominance of small cells.