Jabalpur is the city with a large number of ponds and river tributaries which are the main source of water to drink. The study was needed for the quality of this water. The present study was done to observe the bacteriological quality of water in Jabalpur on the basis of isolation and characterization of E.coli. A total of 117 samples were collected from different water sources in Jabalpur city, comprising 20 each from different banks of river Narmada and public taps, 21 from tube wells, 35 from different ponds, 21 from hand pumps. The result showed an overall 15.38% prevalence of E.coli with sample wise prevalence of 00.00%, 4.76%, 10.00%, 15.00%, 34.00%, and 28.00% in tube wells, hand pumps, public taps, river Narmada and ponds, respectively. Characterization of E.coli with PCR revealed the presence of 83.33% uspA genes. Most of the samples collected ware having the presence of E.coli contamination.