Run-of-river (ROR) dams, often perceived as having minimal environmental impact, can induce significant hydrodynamic changes that alter aquatic ecosystems. We investigated the impacts of an ROR dam on the Madeira River, the largest Amazon tributary, focusing on phytoplankton communities, their ecological implications, and related environmental factors. Our study examined changes in biomass and environmental factors (using General Linear Mixed Models - GLMM), species composition (using PERMANOVA) before and after damming, in both the main channel and tributaries (N = 549 samples). We also identified indicator species associated with different damming phases and regions through an indicator value analysis. The results showed that, following dam construction, the phytoplankton community changed in both the main channel and tributaries, with a shift from lotic diatoms to lentic phytoflagellates. This transition was likely facilitated by altered hydrodynamics and possibly influenced by the decomposition of flooded vegetation in the dam's influence zone. The decomposition of this vegetation could explain both the observed increase in oxygen consumption and the subsequent rise in phytoflagellate biomass after damming. However, despite the overall increase in phytoplankton biomass, the values remained within oligotrophic to mesotrophic conditions, consistent with the low nutrient concentrations recorded. However, we caution that the dam-created hydrodynamic conditions are optimal for phytoplankton growth, potentially exacerbating nutrient-related issues in the future. We recommend proactive management strategies to prevent nutrient enrichment from activities such as agriculture and livestock in isolated Amazon areas affected by dams, thereby mitigating potential degradation of water quality linked to increased phytoplankton biomass.