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Large rivers serve as hotspots for the biogeochemical cycling of nutrients in which the role of microorganisms is crucial. Therefore, understanding the bacterial abundance, diversity, community composition, biogeography, and controlling environmental factors in such habitats is important. However, the information is still unexplored in Himalayan Rivers and their associated ecosystem towards the southern slopes, Nepal. Thus, composition and diversity of particle-attached (PA) and free-living (FL) bacteria was investigated along the mainstream and major tributaries of Koshi River i.e. the largest river of Nepal, using 16S rRNA gene-based Illumina MiSeq platform. Bacterial community composition significantly differed in habitat (mainstream and tributaries) and lifestyle (PA and FL), and the habitat variation was more pronounced than lifestyle variation. Likewise, bacterial communities were dissimilar in more geographically distant sites and vice versa. Distinct dominant taxa in mainstream and tributaries were indicative of different input sources and environmental conditions. Ten OTUs belonging to the phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes contributed to more 50% of the bacterial community dissimilarity in two habitats. Geographical distance and water physicochemical properties explained for the most variation in PA and FL bacteria, respectively. Nonetheless, the individual effect of geographical distance was highest in both PA and FL bacteria. Effect of particulate environmental factor like TSS in variation of PA bacteria and that of dissolved environment factors like DOC and EC in FL bacteria was remarkable.