Investigating drinking water quality is crucial for public health, as clean water access is a fundamental requirement for a healthy life. To comprehensively assess Karachi’s drinking water quality, 152 water samples were systematically collected from five districts: Malir, Karachi West, Karachi East, Karachi South, and Karachi Central. The investigation involved analyzing various physicochemical and microbiological parameters in comparison to WHO 2011 guidelines. Additionally, integrated techniques like principal component analysis and water quality index computation offer insights into both potable and non-potable water aspects, with a focus on health-related well-being. Elevated levels of turbidity and chloride were identified across all five districts, with the residual chloride surpassing permissible limits in Karachi West and Karachi Central. Coliform and E. coli levels exhibited significant variations, with the highest mean values recorded in Karachi Central and the lowest in Karachi East. The overall analysis revealed that only 16.67%, 38.71%, 50%, 43.33%, and 58.06% of the water supply is suitable for drinking in Malir, Karachi West, Karachi East, Karachi South, and Karachi Central, respectively, while 83.33%, 61.29%, 50%, 56.67%, and 41.94% are unsuitable for drinking, posing substantial health risks. Urgent interventions in water quality management and public health are imperative to mitigate these risks associated with substandard drinking water.