Aim This research aimed to monitor pharmaceutical water system by sampling water from all treatment stages, identify bacterial isolates from each phase and determine the most suitable methods to control them. Methods and Results Water samples were collected and examined from pharmaceutical water system in a pharmaceutical factory in Giza, Egypt during 12 months, once per month (from December 2017 to November 2018) from 15 points covering all stages of the treatment process starting from wells, pre‐treatment points; treatment points ending with purified points which are the main source of water used in all pharmaceutical process. In all, 216 water samples were collected and examined, 156 isolates were selected according to morphological characteristics. VITEK system 2 (BioMérieux) was used for identification of all isolates resulting in 24 different identified bacteria. Antibiotic assay test using disc diffusion methods were carried out using seven antibiotics from different groups. Several disinfectants were also examined for efficacy against the isolates to control micro‐organisms in water treatment stage and manufacturing area. The effect of different preservatives (parabens, acids and alcohols) in various pharmaceutical formulas was also tested on bacterial isolates, 63% of formulas were effective against all bacterial isolates. Conclusion Ciprofloxacin was the most effective antibiotic, mixture of 0·45% peracetic acid plus 2·2% of hydrogen peroxide (Minncare 1%) was maximally effective disinfectant, and Cronobacter sakazakii was the most resistant micro‐organism against 22·7% of tested preservatives. Significance and Impact of the study Controlling pharmaceutical manufacturing operation from pathogenic bacteria that affect the quality of drugs.