Chemical and isotopic tracers are used to identify and discriminate the factors controlling the chemical characteristics of surface water in the Andarax River. Along the river, two areas are found to have distinct processes. In the Upper Andarax zone, the surface water (163–645 μS cm−1) is influenced by the direct infiltration associated with precipitations and groundwater discharge from the carbonate aquifer of Sierra de Gádor. This is responsible for the increase in the Sr/Ca and Mg/HCO3 ratio in the surface flow direction. The influence of groundwater on the surface waters contributes to the maintenance of the channel and affects the hydrochemical and isotopic characteristics of the surface water in the area. All of this has been characterized through the combined use of the 234U/238U isotopic ratio (1.59–1.92) and the uranium content (<0.009 μmol/L). In the Middle Andarax, the surface water has an increased salinity (724–1850 μS cm−1) and the values of the Ca/SO4 (0.73–1.38) and Sr/SO4 (0.005–0.008) molar ratios are lower than those of the Upper Andarax zone (1.49–4.17) and (0.009–0.011). This middle zone has an area of temporary water having low B/Li molar ratio (0.31–0.93) values, and another area of permanent flow having similar values (1.07–1.26) to those of the nearby groundwater (1.22–1.26). The groundwater-surface water interaction and the lithological influence notably condition these differences in hydrochemistry. Therefore, the use of hydrochemical and isotopic tracers assists in the understanding of the processes affecting the evolution of the surface waters, considering the interaction with groundwater, the lithology of the aquifers and rocks of the basin, and other relevant environmental factors. This knowledge is useful for the management of water resources in semiarid zones having problems with quality and quantity of surface waters.