Quality of drinking water is one of the greatest anxiety of water consumers with contamination of different pathogenic microorganisms especially fungi and bacteria. Water-borne pathogen contamination in water resources and related diseases are a major water quality concern throughout the world. A motivation for this study was the numerous reports about these contaminations of drinking water and the associated diseases. Water samples were collected from various locations of Ahmedabad in February 2017. The samples were analyzed by 16S rDNA PCR and ITS rDNA PCR since the DNA sequence polymorphisms in the 16S ribosomal DNA and ITS rDNA can be used for the identification of bacterial and fungal species respectively. The PCR amplicons were obtained for each water samples, were sequenced and the species identity was determined using NCBI-BLAST. Bacterial species of Aeromonas, Citrobacter and E. coli along with two fungal species were obtained from municipal water supplies and parab waters. Species of Aspergillus and Penicillium represented the most common fungi in municipal water supplies. This is the first report of molecular study from drinking water facilities of Ahmedabad. This study has clearly revealed that currently, the microbiological quality of the water is inferior for drinking purpose due to bacterial and fungal contamination. This study also showed the potential PCR based identification as a rapid technique for identifying microorganisms in drinking water samples.