Purpose of the research work was to appraise the purified water quality by microbial analysis used in various manufacture in pharmaceutical industry. To have better control water systems samples are collected at regular intervals from the predefined user points. Water samples are checked for Total Aerobic Microbial Count (TAMC), Total Yeast and Mold Count (TYMC), Escherichia coli, Salmonella species, Staphylococcus aureus, Pseudomonas aeruginosa, Out of 2424 samples tested, 93.3% are positive, 6.6% are negative, 0.1% out of alert and no growth observed for TYMC, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Salmonella sp. Investigation performed for 0.1% (4 samples) for which out of alert limit observed and identified the microbes such as Ralstonia solanacearum, Ralstonia mannitolilytica, Bacterium Sanya and Sphingomonas sp. based on the molecular characterization Based on the outcome of the results RO membrane replaced and increased the sanitization frequency. Further water samples were monitored as per the predefined frequency and observed that the results are within acceptance criteria.