Free-living amoebae (FLA) are widely distributed in the environment. Among these, Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Vermamoeba vermiformis have been reported as human pathogens with health effects ranging from lethal encephalitis to different epithelial disorders. Despite this, FLA still present many diagnostic challenges. The aim of this study was to develop a rapid and efficient multiplex real-time quantitative polymerase chain reaction (qPCR) to simultaneously detect Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis in different water sources. For the validation of the qPCR assay, 38 samples (19 tap water and 19 stagnant water sources) were analyzed. The qPCR assay accurately identified the four types of FLA with no cross-reactivity. Considering water samples with results subsequently confirmed by conventional PCR, the multiplex qPCR assay detected 18/38 (47.4%) positive samples (Acanthamoeba spp. in 44.7% and V. vermiformis in 31.6%) and growth in nonnutritive agar (NNA) cultures identified 7/38 (18.4%) positive samples. Of the tap water samples analyzed, 26.3% of samples positive for FLA were detected by growth in NNA culture whereas 31.6% were identified by qPCR. In addition, FLA were detected in 2/19 stagnant water samples (10.5%) by growth in NNA culture and in 12/19 stagnant water samples (63.2%) by qPCR. Neither N. fowleri nor B. mandrillaris was detected in the water samples analyzed. In conclusion, the qPCR developed showed its potential as a rapid tool for detection of Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis. Moreover, FLA species were detected in half of the water sources evaluated, suggesting the importance of the surveillance of these potential infectious agents.