Acanthamoeba species are ubiquitous, free-living organisms found in the environment. They can cause a sight-threatening cornea disease, termed Acanthamoeba keratitis, and are often misdiagnosed, causing delayed administration of the correct treatment. Herein, we report a case of Acanthamoeba keratitis diagnosed without culture. A 12-year-old girl with a history of wearing contact lenses presented with complaints of pain, irritation, and hyperemia in the left eye. Corneal scraping-smeared slide, and liquids with contact lenses were submitted to the clinical microbiology laboratory. Cultures of Acanthamoeba spp. were not available; thus, they were stained with calcofluor white. The isolation of Acanthamoeba from the corneal scraping allowed the detection of trophozoites and cysts based on their morphological characteristics. PCR targeting the 18s rRNA gene and subsequent sequencing revealed 99% identity with the Acanthamoeba spp. Although it is challenging to find real-world evidence of Acanthamoeba in clinical microbiology without using culture methods, this case underscores the need for clinical microbiology laboratories to maintain their inspection capabilities.