<p>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 tends to change in response to the new conditions that 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 richness of phytoplankton classes was higher during the pre-dam period, the dry season, and in the river’s main channel. The predominant classes 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 the most common and abundant ones, suggesting that the damming of the Tocantins River led to the loss of sensitive and specialist species.</p>