Acanthamoeba can cause visually destructive Acanthamoeba keratitis (AK) in contact lens (CL) users. The purpose of this study was to determine whether Acanthamoeba was present in the CL cases of CL wearers and to develop techniques to prevent the contaminations. To accomplish this, 512 CL case samples were collected from 305 healthy CL wearers. Using real-time PCR, Acanthamoeba DNA was detected in 19.1% of CL cases, however their presence was not directly associated with poor CL case care. Instead, the presence of Acanthamoeba DNA was associated with significant levels of many different bacterial species. When the CL cases underwent metagenomic analysis, the most abundant bacterial orders were Enterobacteriales followed by Burkholderiales, Pseudomonadales, and Flavobacteriales. The presence of Acanthamoeba was characterized by Propionibacterium acnes and Rothia aeria and was also associated with an increase in the α diversity. Collectively, Acanthamoeba contamination occurs when a diversified bacterial flora is present in CL cases. This can effectively be prevented by careful and thorough CL case care.