South Africa is facing a water crisis with the available surface water resources almost fully allocated to meet socio-economic and population growth. Rural industrial development has led to headwater catchments becoming mosaics of multiple anthropogenic activities, with compounding and synergistic land-use stressors often undermining adjacent river water quality and ecosystem functionality. Although water quality monitoring remains critical for water resource management, it has limited utility in untangling sources of ecosystem stress, as many of the tracers (e.g. nutrients) have both natural and anthropogenic sources. Stable isotopes of δ13C, δ15N and δ34S have the dual ability to trace sources of aquatic ecosystem stress and ascertain ecosystem functionality, but have not been widely assessed together in the South African context, particularly in conjunction with additional physico-chemical water quality tracers. This research investigated δ13C, δ15N and δ34S of aquatic tissues in two rivers across a land-use intensity gradient within Gwalthe catchment, North-West Province. The δ13C was a key indicator of anaerobic bacterial chemoautotrophy following its depletion within deep water reservoirs. The δ15N became enriched after agricultural and urban activities, accompanied by elevated ammonium and phosphate concentrations. The δ15N values did, however, become significantly depleted following illicit mining activities. δ34S was a key indicator for mining stress, due to isotopically depleted sulfur entering the food web from chrome and platinum mine dumps. Our findings indicate that δ13C, δ15N, and δ34S from the tissues of aquatic organism together with physico-chemical water quality tracers can provide a tool to discriminate between key land-use stressors.