Water quality of the sacred River Bhagirathi was evaluated by microbiological and physico-chemical characteristics of water. Monthly water samples were collected from the upper zone (1,158–4,100 m a.s.l.) to lower zone (457–1,158 m a.s.l.) of the river for a period of two years during October, 2013 to September, 2015. The data on microbial density revealed that CFU count was minimum (13,185 CFU. ml−1) in the winter season and maximum (36,410 CFU.ml−1) in the monsoon season, when the degradation of water quality was maximum due to mixing of a large amount of allochthonous materials from the catchment area. No total coliform (TC) and fecal coliform (FC) was found in the water samples from Gaumukh (4,100 m) to Gangotri (3,140 m) during the two-year sampling period due to minimum anthropogenic pressure. However, total coliform and fecal coliform were recorded downstream of Gangotri (TC: 980 and FC: 120) and Harshil (TC: 1,100 and FC: 200). Microbial density was recorded to be high in the lower stretch: Uttarkashi (TC: 2,850 and FC: 860) Tehri (TC: 5,000 and FC: 4,200), and Deoprayag (TC: 3,800 and FC: 2,700). A total of 14 bacterial, 4 Actinomycetes, and 11 fungal species were found in the Bhagirathi River.