Aim of the Study: The aim was to investigate the bacteriological quality of drinking water, and explore the factors involved in the knowledge of the public about the quality of drinking water in Najran region, Saudi Arabia. Study Design: A cross-sectional descriptive study. Materials and Methods: A total of 160 water samples were collected. Total coliforms, fecal coliform, and fecal streptococci were counted using Most Probable Number method. The bacterial genes lacZ and uidA specific to total coliforms and Escherichia coli, respectively, were detected using multiplex polymerase chain reaction. An interview was conducted with 1200 residents using a questionnaire. Results: Total coliforms were detected in 8 (20%) of 40 samples from wells, 13 (32.5%) of 40 samples from tankers, and 55 (68.8%) of 80 samples from roof tanks. Twenty (25%) and 8 (10%) samples from roof tanks were positive for E. coli and Streptococcus faecalis, respectively. Of the 1200 residents participating in the study, 10%, 45.5%, and 44.5% claimed that they depended on municipal water, bottled water, and well water, respectively. The majority (95.5%) reported the use of roof water tanks as a source of water supply in their homes. Most people (80%) believed that drinking water transmitted diseases. However, only 25% of them participated in educational programs on the effect of polluted water on health. Conclusions: Our results could help health authorities consider a proper regular monitoring program and a sustainable continuous assessment of the quality of well water. In addition, this study highlights the importance of the awareness and educational programs for residents on the effect of polluted water on public health.