The decline of freshwater ecosystems, generally result from land use activities in the river catchment and is of great concern worldwide. This study was conducted along Umtata River in the Eastern Cape province of South Africa between May 2010 and April 2011. The study was aimed at identifying macrophytes families (to species level) and determining the stable isotope signatures (C:N ratios, δ13C and δ15N) and to relate their isotopic signatures to the land use activities along the river catchment. Analysis of variance was performed to test the effect of sites and sampling period on the C:N ratios, δ13C and δ15N signatures. There were 16 macrophyte families represented by 26 species recorded along the river. There was only a significant difference in sites and sampling period in δ15N (p< 0.05). The highest C:N ratios value (30.75±9.65‰) was recorded in the upper reaches while the lowest value (6.10±2.35‰) occurred in the lower reaches. The δ13C values varied throughout the length of the river with highest values (-19.63±5.44‰) obtained in the middle reaches. Spatial variation was evident in δ15N throughout the length of the river and showed increase from the upper reaches to middle reaches and decreased towards lower reaches. The δ15N ranged from 3.92±2.43‰ in the upper reaches to 10.02±4.56‰ in the middle reaches. Temporal variation in δ15N was also evident throughout the sampling period with highest peak in May (9.77± 4.49‰) and lowest in February (0.50±2.49‰) respectively. The highest values of isotope signatures at spatial level demonstrated the true reflection of urban development, sewage discharge and agricultural activities taking place along the river system. Continued monitoring is recommended that may ultimately come up with a better management options for the communities living within the study area, and also to better enhanced land utilization.