ABSTRACT Biological assessment of rivers is critical for evaluating ecosystem health, detecting ecological changes, validating physicochemical monitoring data, assessing the effectiveness of water treatment systems, conserving biodiversity, and guiding water resource management. In this study, the surface water quality of the Karkheh River basin was evaluated using the National Sanitation Foundation Water Quality Index (NSFWQI) and the Iranian Surface Water Quality Index (IRWQIsc) for qualitative analysis. For biological monitoring, the Biological Monitoring Working Party (BMWP) index, along with the Average Score per Taxon (ASPT) and the Hilsenhoff Biotic Index (HFBI), was employed to provide a comprehensive assessment. Water quality was analyzed across 18 sampling stations, revealing significant spatial variation. Upstream areas exhibited relatively good conditions, with NSFWQI and IRWQIsc values of 59 and 68, respectively. In contrast, downstream locations near urban and agricultural areas demonstrated poor water quality, with values of 27 and 33, respectively. Overall, approximately 70% of the river's length has been severely impacted, highlighting the need for targeted planning and management to mitigate pollution. The biological indices corroborated the physicochemical results, revealing severe ecological stress in polluted regions. The close alignment between the NSFWQI and biological indicators emphasizes the importance of a multifaceted approach in assessing aquatic health. Key factors contributing to the river's degradation include excessive water extraction, untreated wastewater discharge, and diminished self-purification capacity, particularly in downstream areas.