Mercury is a metal present in the Earth’s crust, but due to human contribution, its concentration can increase, causing environmental impacts to aquatic ecosystems, among others. The Reis Magos River Hydrographic Basin represents economic and socio-environmental importance for the state of Espírito Santo, Brazil. However, there are not many publications regarding the quality of water and sediments, so no data is reported concerning the total concentration of Hg. Thus, the present work aimed to evaluate the distribution of total Hg in water and sediments along this hydrographic basin. For a better inference, physicochemical parameters of the water were determined (temperature, pH, electrical conductivity, oxidation-reduction potential (ORP), turbidity, dissolved oxygen (DO), total dissolved solids (TDS), and salinity), and in the sediments, the contents of matter organic matter, pH, carbonates and granulometry. Mercury determination was performed by Thermodecomposition and Amalgamation Atomic Absorption Spectrometry (TDA AAS) with a DMA-80 spectrometer. The Hg determined in the water was lower than the limit of quantification, 0.14 µg∙L−1, which is lower than the maximum limits recommended by world reference environmental agencies. In the sediment samples, the Hg found were below 170 µg∙kg−1, values below which there is less possibility of an adverse effect on the biota. However, when the degree of anthropic contribution was evaluated using the Geoaccumulation index (IGeo), the contamination factor (CF), and the ecological risk potential index (EF), there was evidence of moderate pollution. Thus, this highlighted the need for monitoring the region since climatic variations and physical-chemical parameters influence the redistribution of Hg between the water/sediment interface.