The degradation of water resources available for human consumption is increasing with the continuous release of chemicals into aquatic environments and their inefficient removal in wastewater treatment. Several watersheds in Brazil, such as the Iguaçu River, are impacted by multiple sources of pollution and lack information about their pollution status. The Iguaçu River Basin has great socioeconomic and environmental relevance both for the supply of water resources and its high hydroelectric potential, as well as for the high rate of endemism of its ichthyofauna. Also, it is home to large conservation units, such as the Iguaçu National Park, recognized by Unesco as a Natural Heritage of Humanity. Thus, the current review discusses the chemical pollution in the Iguaçu River Basin (IRB) approaching: i) the main sources of pollution; ii) the occurrence of inorganic and organic micropollutants; iii) the available ecotoxicological data, and iv) the socioeconomic impacts in three regions of the upper, middle, and lower IRB. Different studies reported relevant levels of emerging contaminants, persistent organic pollutants (POPs), toxic metals, and polycyclic aromatic hydrocarbons (PAHs), detected in the water and sediment samples, especially in the upper IRB region, associated with domestic and industrial effluents. Additionally, significant concentrations of pesticides and toxic metals were also detected in the lower IRB, revealing that agricultural practices are also relevant sources of chemicals for this watershed. More recently, studies indicated an association between fish pathologies and the detection of micropollutants in the water and sediments from IRB. The identification of the main sources of pollutants, associated with the distribution of hazardous chemicals in the IRB and their potential effects on the biota, as described in this review, represents an important strategy to support water management by public authorities for reducing potential risks of the local endemic biodiversity and exposed human populations.