The Karun River, western Iran, is the largest (catchment area of 57’059 km2) and the only navigable river system in Iran. It flows westwards out of the Zagros mountain range (max. altitude 4409 m.a.s.l.), traverses the Khuzestan plain, and joins the Shatt al-Arab, which then enters the Persian Gulf. The freshwater ecosystems of the Karun are affected by various uses of its water and catchment (i.e. agriculture, rural development, aquaculture and hydropower facilities) but the impacts of these activities on fish and macroinvertebrate communities is largely unknown. This is particularly problematic because the Zagros mountain range is the heart of the Irano-Anatolian Biodiversity Hotspot. As part of a collaborative project between Iranian and Swiss research groups, we investigated fish and macroinvertebrate communities and the abiotic conditions at 53 lotic sites in the entire river system in spring and summer 2019. Sites ranged from small high-altitude creeks to large lowland rivers, the latter often affected by various anthropogenic pressures. Pristine abiotic conditions were mainly restricted to small high-altitude sites, which had low chemical pollution and excellent habitat features (in particular with low fine sediment deposition, natural channel morphology and intact riparian vegetation). Fishes were sampled in all habitats of the respective sites by backpack electro fishing and benthic macroinvertebrates on ten transects per site using kicknetting and surber sampling. Samples were preserved in formaldehyde and transferred to the laboratory for identification. We identified 36 fish species and 77 macroinvertebrate families. Fish communities were dominated by cyprinids (65.2%; mainly Capoeta coadi, Garra rufa, Capoeta aculeata) and macroinvertebrate communities by chironomids (46.4%), both taxa being relatively insensitive to habitat degradation. Overall, we observed significant correlations between fish and macroinvertebrate communities in terms of various diversity indices (based on species richness and evenness) and overall abundance, suggesting similar community responses of both organism groups to the anthropogenic and natural gradients in the Karun. Specifically, we measured highest fish diversity in large lowland river reaches despite being rather polluted, thus pointing at positive effects of connectivity and habitat heterogeneity, but probably also of introductions, for biodiversity. Rare endemic fish species (e.g. benthic species such as Sasanidus kermanshahensis, Oxynoemacheilus freyhofi, Turcinoemacheilus hafezi and Turcinoemacheilus saadii) were restricted to relatively clean headwater sites characterized by coarse sediment and high flow velocity. We measured highest macroinvertebrate diversity in sites with excellent habitat features albeit moderate chemical quality. In particular, we observed highest EPT diversity (the orders Ephemeroptera, Plecoptera, Trichoptera, comprising many sensitive species) in riffle sites characterized by cobble sediment and high flow velocity. Findings from our project will contribute to the management and conservation of freshwater biodiversity in this region, which currently undergoes major environmental changes due to human population growth, increasing resource demands and climate change.