Abstract. Abstract Aim The present study investigated the influence of environmental variables on macrobenthic fauna in the Achenkovil River, Southern Western Ghats, Kerala. The knowledge of aquatic ecosystems in many remote areas is very scanty and no one has yet made an attempt to study the macrobenthic fauna of Achenkovil river. Since, macrobenthos play an important role in the benthic community structure a better knowledge of their ecology and distributional patterns in remote freshwater ecosystems would be of great interest for a better understanding of their functioning. Methods A bimonthly sampling was conducted from February 2019 to January 2020. Water samples for physicochemical analysis were carried to the laboratory in clean polyethylene bottles. The macrobenthic fauna was collected using Van Veen grab (0.025m2). Multivariate statistical technique such as Principal Component Analysis was used to monitor the variation in environmental parameters. The diversity of macrobenthic fauna was studied using Simpson_1-D, Shannon_H, Evenness_e^H/S, and Margalef’s indices. We analyzed the relationship between environmental variables and macrobenthic fauna, using Canonical Correlation Analysis and Pearson correlation. Results A total of 3563 individuals belonging to 9 orders, 32 families, 32 genera, and 32 species were identified. The most dominant order was Ephemeroptera followed by Coleoptera and Trichoptera and the least dominant was Zygoptera. The maximum number of macrobenthic fauna was observed in station 1(S1), (1461 Ind/m2) and the least in station 9 (S9), (37 Ind/m2). Station 1 (S1) is a pristine forest area, so this area is considered the reference site. The macrobenthic fauna in the pristine headwater forests (S1) was much more diverse than in the river mouth (S9) which is a highly impacted region. This region is facing various kinds of anthropogenic stress resulting in pollution and deterioration of water quality. The macrobenthic community structure in the Achenkovil River was significantly associated to the variations in water quality due to various natural as well as anthropogenic stresses. Conclusions It can be concluded that Ephemeroptera, Coleoptera, and Trichoptera were the most dominant groups in the undisturbed habitats in the river during the study period. The predominance of Chironomidae species, especially from the Chironomus genus in the midstream and downstream segments of the river is possibly due to their ability to adapt to various environmental conditions or habitats and their tolerance to the low oxygen content in anoxic conditions of the bottom sediment. The lowest abundance of macrobenthos at all the studied stations was observed at station 9 (S9) during dry seasons. S9 is a highly impacted region, characterized by high temperature, turbidity, TDS, pH, conductivity, nitrate, and phosphate values. For this reason, anthropogenic stress and changing environmental conditions are the main factors affecting the distribution of the macrobenthic fauna of the Achenkovil River basin, Kerala.