Acanthamoeba, human pathogens and natural hosts of pathogenic bacteria, may be accurately detected and quantified by real-time PCR if Acanthamoeba DNA are properly extracted and PCR inhibitors are effectively eliminated. However, the optimization of DNA extraction methods has not been reported for Acanthamoeba. This study compared the effectiveness of two DNA extraction/purification methods (FastDNA Spin Kit for soil and Wizard SV genomic DNA Purification System) by using trophozoites and cysts of Acanthamoeba castellanii and water and biofilm samples of cooling towers. DNA of A. castellanii extracted with the FastDNA Kit and quantified by TaqMan PCR resulted in a lower variation (CV of Ct < 3%), greater linearity (R2=0.99), and higher slopes (1.177–1.187 log fg DNA/log cell number) as compared to that by the Wizard Kit. For field testing, the number of Acanthamoeba-positive samples and the Acanthamoeba DNA quantity were both greater with the FastDNA Kit than with the Wizard Kit (P=0.016 and <0.0001, respectively). Beneficial effects with dilutions of extracted DNA were also revealed with the FastDNA Kit (P=0.0003). In conclusion, DNA extraction by the FastDNA Kit coupled with dilution of extracted DNA and PCR analysis are recommended for detecting and quantifying environmental Acanthamoeba.