The contamination of processed or unprocessed drinking water by pathogenic bacteria has been reported worldwide. Even though a high incidence of waterborne diseases have been well documented, enterobacteriaceae is an under acknowledged pathogen of concern to public health in India. The study was aimed to isolate and biochemically characterize Enterobacteriaceae as well as to determine  antibiotic sensitivity pattern and primer designing specific to their signature gene sequences for rapid detection by PCR methods. Total plate count was determined in potable water sample collected from three different sites of Jaipur city and was found to be nearly equal or more than 105 cfu/ ml. Enterobacteriaceae were enumerated using the most probable number method (MPN index). Enterobacteriaceae (n = 36), randomly isolated from three sites, were identified, biochemically characterized and were screened for sensitivity to 9 antimicrobials by the disk diffusion method. The isolated bacteria had shown variable susceptibility and resistance patterns in response to different antibiotics used in the study. Polymerase chain reaction based methods can be a powerful molecular technique for rapid, sensitive and specific detection. Hence, in the present study primers were also designed specific to virulence signature sequences and their specificity was assessed by BLAST.