Understanding the impact of human activities on fish communities is critical for the effective conservation of Amazonian ecosystems. In this study, we sampled environmental DNA (eDNA) from 17 sites (lakes and rivers) in two Peruvian national reserves, Pacaya-Samiria and Allpahuayo-Mishana, using a metabarcoding approach with four genetic markers (2 COI and 2 12S). We hypothesized that fish community composition and functional diversity in Amazonian freshwater systems could be impacted by anthropogenic factors, such as deforestation or a set of variables included in the Human Modification Index (SEDAC). Our analyses identified 319 species in Pacaya-Samiria and 207 species in Allpahuayo-Mishana, demonstrating the efficacy of eDNA for generating robust biodiversity inventories. While human disturbances did not significantly reduce species richness, they altered species composition and impacted functional diversity. Specifically, deforestation reduced functional richness, indicating the loss of ecological functions, while increasing functional dispersion, reflecting shifts towards more generalist species. These findings underline the vulnerability of functional diversity to anthropogenic pressures, even in high-biodiversity systems, and emphasize the need for conservation strategies that incorporate functional diversity alongside taxonomic diversity. In practical terms, management actions should prioritize the maintenance of structurally complex and forested riparian habitats, which support functionally diverse fish assemblages, and monitor functional traits as early indicators of ecosystem degradation. Approaches such as functional vulnerability analysis can help identify key ecological functions at risk, while targeted habitat restoration in deforested buffer zones may mitigate human impacts. Strengthening community-based management and integrating eDNA monitoring into long-term conservation plans offer a proactive, adaptable framework for safeguarding Amazonian freshwater ecosystems.