The Ganges is the largest river of India, worshiped by Hindus with a belief that bathing in the river causes the remission of sins and is considered very pure. It is heavily polluted by the unrestricted human usage including ritual practices, urbanization, and industrialization. Such perturbations may subsequently influence the bacterial community composition and ecosystem functioning. Here, we applied targeted amplicon sequencing to determine the impact of spatial variation on the microbial community assemblage of the Ganga River. The river bacterial community demonstrates taxonomic variability across the sites with accumulation of Firmicutes (20.9%) Verrucomicrobia (6.09%), Actinobacteria (4.51%), and Synergistetes (1.16%), at rural site while Proteobacteria (49.4%) and Bacteroidetes (12.7%) predominate at urban sites. Furthermore, sites under study establish the unique taxonomic signature which could represent the impact of spatial variation on the microbial community assemblage.