Studying agricultural pressure effects on the functional composition of aquatic communities (e.g., trophic organisation) is critical to understanding how anthropogenic influence ecosystem functions and services. Studies and tools for investigating the impact of human activities on the functional organisation of macroinvertebrates in tropical streams remain scarce, despite Africa witnessing the most significant increase in poorly managed agricultural practices worldwide. We investigated the responses of Ephemeroptera, Plecoptera and Trichoptera (EPTs) functional feeding groups (FFGs) to agricultural pollution in the Kat River, Eastern Cape, South Africa, to identify potential FFG indicators for long-term monitoring of agricultural pollution in the region. Physicochemical parameters and macroinvertebrates were sampled from eight sites delineated into four land-use impact categories (highly impacted, HIC; impacted category, IC; moderately impacted, MIC and least impacted, LIC) based on the percentage of agricultural land cover. Our results revealed land use and season-influenced FFGs in the river, with shredder abundances decreasing with increasing agricultural stress. Conversely, collectors, predators and scrapers were tolerant of agricultural disturbance. Nutrient variables (NO3-N, NO2-N and NH4-N), canopy and depth, were the critical variables that affected FFG structure in the river. Our findings provided more understanding and insights into the EPT functional structure in relation to agricultural pollution and can contribute to the development of trait-based biomonitoring tools in Africa.