Access to safe water and sanitation is a critical global challenge, posing significant health risks worldwide due to waterborne diseases. This study investigates the efficacy of ultraviolet type C radiation as a disinfection method for improving water quality. The research elucidates UV-C’s mechanism of action, highlighting its ability to disrupt DNA and RNA replication, thereby inactivating pathogens. Furthermore, the study analyses the influence of key factors on UV-C disinfection effectiveness, including water turbidity and the presence of dissolved organic matter, which can attenuate UV-C penetration and reduce treatment efficiency. The experimental results demonstrate a substantial reduction in microbial content following UV-C treatment. River water samples exhibited a 57.143% reduction in microbial load, while well water samples showed a 50% reduction. Notably, Escherichia coli (E. coli) concentrations decreased significantly, with an 83.33% reduction in well water and a 62.5% reduction in borehole water. This study makes a novel contribution to the understanding of UV-C disinfection by identifying the presence of resistant organisms, including Adenoviruses, Bacterial spores, and the Protozoan Acanthamoeba, in water samples. This finding expands the scope of UV-C research beyond easily culturable bacteria. To address this challenge, future investigations should explore synergistic disinfection strategies, such as combining UV-C treatment with advanced oxidation processes. Optimising UV-C system designs and developing robust, real-time monitoring systems capable of detecting and quantifying known and emerging UV-resistant pathogens are crucial for ensuring comprehensive water decontamination.