<p>Traditional monitoring approaches focus solely on the physicochemical characterisation of water. Macroinvertebrate-based biomonitoring of water bodies for ecosystem health has emerged as a cost-effective, holistic, and accurate method. Biomonitoring helps capture the cumulative impacts of stressors on aquatic ecosystems. Macroinvertebrates have been widely used as indicators across North America, Europe, and Australia. In South Asia, their use has been limited due to inadequate validation of methods in local ecological and hydrological contexts. In this study, the Cauvery River Basin (CRB) in Karnataka, India, was selected, including the mainstream and its tributaries. Sampling was conducted in the pre-monsoon (April–May) and post-monsoon (October–November) seasons of 2025. A total of 36 macroinvertebrate families were recorded across the basin. The ecosystem health of different water bodies was first assessed using a rapid bioassessment technique and later validated using macroinvertebrate-based scoring and index methods, i.e., Biological Monitoring Working Party Score (BMWP), Ganga River System Biotic Score (GRSbios), Hindu Kush–Himalaya Biotic Score (HKHbios), and Family Biotic Index (FBI). The benthic macroinvertebrate communities in the basin are dominated by Mollusca (both gastropods and bivalves), followed by insects and annelids. Comparison of findings during method validation revealed promising results: GRSbios closely matched the pre-classified status, whereas other methods either slightly underestimated or overestimated the health status. It is recommended that more sites be studied across different seasons and sub-basins with varying ecosystem health statuses in the CRB, and that the GRSbios list of indicator species with tolerance values be enriched, leading to the development of a dedicated biotic index for Peninsular India.</p>