Acanthamoeba spp. are known to cause human diseases such as granulomatous amoebic encephalitis and keratitis. These protozoa can persist in aquatic environments for extended periods and exhibit resistance to disinfectants used in water treatment, posing a significant threat to public health. Traditional culture and molecular methods have limitations in the rapid detection of viable Acanthamoeba in water. Therefore, we optimized real-time PCR (qPCR) combined with propidium monoazide (PMA) and evaluated the applicability of PMA-qPCR for the rapid detection of viable Acanthamoeba throughout the tap water production process. Using a PMA concentration of 50 µM, we successfully differentiated viable from non-viable cells and selected a 180-bp fragment of the 18S rRNA gene to enhance qPCR efficiency while minimizing amplification from non-viable cells. Application of PMA-qPCR to samples collected from each stage of the water treatment process, from raw water to purified water, enabled the detection of viable Acanthamoeba in samples with turbidity levels between 0.03 and 13.7 nephelometric turbidity units. These results demonstrate that PMA-qPCR is a suitable tool for monitoring pathogenic Acanthamoeba in drinking water supplies and enabling prompt responses to potential waterborne contamination events.