Objective To evaluate the diagnostic value of real-time polymerase chain reaction (PCR) for detecting Acanthamoeba in eyes diagnosed with Acanthamoeba keratitis (AK) by conventional tests. In addition, to determine the preoperative prognosis-determining factors in eyes with AK. Design Retrospective, cross-sectional study. Participants A total of 104 eyes of 103 patients who were diagnosed with AK or with bacterial or bacteria-associated keratitis (BK) by conventional tests. Methods Twenty-nine eyes with AK and 75 eyes with BK were evaluated for Acanthamoeba and bacterial DNA by real-time PCR. The Acanthamoeba copy numbers, bacterial load, and clinical parameters in the patients with AK were assessed for those significantly associated with poor outcome, that is, final visual acuity of <20/50 or requiring keratoplasty, by logistic regression analysis. Main Outcome Measures Acanthamoeba DNA copy number, bacterial DNA copy number, and odds ratio (OR) for poor prognosis. Results The detection of amoebic DNA was 50 times more sensitive by real-time PCR than by conventional cyst counting. The Acanthamoeba copy numbers at the first visit (mean: 4.7×105±3.2×105 copies) were significantly correlated with the AK stage, and both were significant risk factors for a poor outcome. The Acanthamoeba DNA copy numbers at the first visit and AK stage had a significantly high risk for poor outcome (OR of Acanthamoeba DNA copy per logarithm of copy numbers: 3.48, 95% confidence interval [CI], 1.04–111.63, P<0.05; OR of AK stage: 2.8 per stage increase, 95% CI, 1.07–7.30, P<0.05, after adjustment of age). In the AK cases with poor outcome, the amoebic DNA was not reduced by more than 90% after 1 month of treatment. The weak amoebic reduction was significantly associated with advanced AK stages or previous use of steroids. Bacterial 16S rDNA was detected in 53.6% of the eyes with AK, but it was not associated with any risk for refractoriness. Conclusions Real-time PCR was effective in detecting and managing AK. The Acanthamoeba copy number and AK stage at the first visit were significantly associated with poor outcome. Financial Disclosure(s) The author(s) have no proprietary or commercial interest in any materials discussed in this article.