In order to evaluate the water quality of one of the most polluted urban river in Malaysia, the Penchala River, performance of eight biotic indices, Biomonitoring Working Party (BMWP), BMWPThai, BMWPViet, Average Score Per Taxon (ASPT), ASPTThai, BMWPViet, Family Biotic Index (FBI), and Singapore Biotic Index (SingScore), was compared. The water quality categorization based on these biotic indices was then compared with the categorization of Malaysian Water Quality Index (WQI) derived from measurements of six water physicochemical parameters (pH, BOD, COD, NH3-N, DO, and TSS). The river was divided into four sections: upstream section (recreational area), middle stream 1 (residential area), middle stream 2 (commercial area), and downstream. Abundance and diversity of the macroinvertebrates were the highest in the upstream section (407 individual and H' = 1.56, respectively), followed by the middle stream 1 (356 individual and H' = 0.82). The least abundance was recorded in the downstream section (214 individual). Among all biotic indices, BMWP was the most reliable in evaluating the water quality of this urban river as their classifications were comparable to the WQI. BMWPs in this study have strong relationships with dissolved oxygen (DO) content. Our results demonstrated that the biotic indices were more sensitive towards organic pollution than the WQI. BMWP indices especially BMWPViet were the most reliable and could be adopted along with the WQI for assessment of water quality in urban rivers.