One effective method for assessing the health of rivers and the impact of human activities on water quality is the use of macrobenthos populations as indicators of pollution. This study investigated the diversity and abundance of macrobenthos in the Karaj River over four months—August, September, October, and November—at six different stations in 2022. Sampling was conducted using a Surber sampler (30 × 35 cm) with three replicates per station, and the samples were transferred to the laboratory for analysis. We calculated several diversity indices, including Simpson and Shannon-Wiener, as well as bioindicators such as the Hilsenhoff Index, BMWP, EPT, dominant family percentage, and species richness for all stations. A total of 14 families of large benthic invertebrates were identified, representing 7 orders and 4 classes, with the Baetidae family from the Ephemeroptera order being dominant across all stations. The highest biodiversity was observed in August. Overall, upstream stations showed higher diversity indices than downstream stations. The dominant family percentage index revealed that the Baetidae family was the most frequent, and approximately 72% of the EPT index was associated with the three upstream stations near the dam. According to the BMWP index, all stations were classified as "very bad" or "severely polluted." The Hilsenhoff Index assessed the Pol-e-Khawab station as "very good" (indicating very low pollution risk), the Asara, Sierra, Vine, and Purkan stations as "good" (indicating moderate organic pollution), and the Adran station as "suitable" (indicating significant organic pollution). The Shannon Index identified all stations as relatively polluted. In conclusion, the Hilsenhoff biological index is a reliable tool for evaluating the water quality of the Karaj River, which, based on this index, is considered to be in an average state of quality.