Yersinia enterocolitica, an important food and water-borne enteropathogen cause acute diarrhoea, terminal ileitis and mesenteric lymphadenitis. This study investigated the occurrence, antibiotic sensitivity and virulence factors of yersinial isolates from drinking water samples from Ludhiana, Punjab, India. A total of 418 drinking water samples from various water utilities were analyzed for occurrence of Y. enterocoliitica and faecal coliforms (Escherichia coli). Y. enterocolitica was detected in 78.09% of Municipal Corporation (MC) drinking water samples, 59.02% Submersible pump drinking water samples and 53.12% of Hand pump samples where as E. coli was found in 53.71% of Municipal Corporation (MC) drinking water samples, 29.16% Submersible pump drinking water samples and 8% of samples from Hand pumps. There was no positive correlation between the simultaneous occurrence of Y. enterocolitica and E. coli (P < 0.005; R-2 = 0.88). All the isolates of Y. enterocolitica were positive for virulence marker test Congo dye uptake. Significant histopathological and ultrastructural alterations in liver, lungs, kidney and intestine were reported in experimentally infected mice. All the isolates (n = 291) from water showed multiple antibiotic resistance (MAR). The MAR indices for Y. enterocolitica isolates is 0.5 (> 0.2). These results highlight the pathogenic potential of Y. enterocolitica which poses a public health concern.