Access to clean, sufficient drinking water is a fundamental human necessity. However, many regions, particularly developing nations, face challenges in providing clean drinking water. This study aimed to investigate the presence of pathogenic bacteria in drinking water samples from Sehore district of Madhya Pradesh, and highlight the importance of rapid detection methods. Water samples were collected from various sources including taps, borewells, water tanks, and the Narmada river across different villages of Sehore district. These samples were cultivated on MacConkey , Cetrimide and Xylose lysine desoxyscholate (XLD) Agar medium and incubated at 37°C till 24 to 48 hours for bacterial growth. Each randomly selected bacterial colony was then subjected to Colony PCR using bacteria-specific primers for the detection of Pseudomonas sp., Escherichia coli, and Salmonella sp. The Colony PCR results confirmed the presence of these pathogenic bacterial species, with high presence of Escherichia coli, moderate prevalence of Pseudomonas sp. while low prevalence recorded for Salmonella sp. The presence of these bacteria could pose significant health risks to the local population. The study also utilized molecular and biochemical techniques to identify Escherichia coli, Pseudomonas sp. and Salmonella spp., The findings indicated varying levels of contamination across different water sources, with certain samples showing higher concentrations of pathogenic bacteria. Furthermore, the study evaluated the effectiveness of rapid detection methods such as polymerase chain reaction (PCR) in quickly identifying pathogenic bacteria in drinking water. These methods proved to be valuable tools for timely detection of bacterial contaminants, underscoring the importance of regular monitoring and swift action to ensure public health and safety.