Reservoirs transform the physical, biological and chemical characteristics of rivers, altering their flow dynamics and the availability of nutrients. After a river is dammed, the species composition in the reservoir and its tributaries tend to change in response to the new conditions which are generated. This work investigates the tributary rivers and sites along the main channel of the Tocantins River’s middle course. The study was conducted between 2009 and 2013 in the pre- and post-dam periods to evaluate the effects of the dam on phytoplankton. The area of study was located along the middle course of the Tocantins River, where the Estreito hydroelectric power plant is found. The variables of ammonium, nitrate and inorganic phosphorous were those that most contributed to the environmental variation post-damming. The richness in classes of phytoplankton was higher during the pre-dam period, the dry season, and in the environment of the river’s main channel. The classes which stood out the most were Bacillariophyceae, Chlorophyceae and Cyanophyceae. We verified that damming the Tocantins River negatively affected the phytoplankton communities, reducing their richness and density in the post-dam period. The results showed that the species with the largest contributions to beta diversity were found to be the most common and abundant ones, suggesting that the damming of the Tocantins River has led to the loss of sensitive and specialist species.