This study correlated confocal microscopic images obtained using the Nidek ConfoScan 2.0 System in corneas with clinical suspicion of Acanthamoeba keratitis, with diagnosis confirmed by either cytological and/or histological analysis. Fifteen eyes of 14 patients with a clinical diagnosis of Acanthamoeba keratitis underwent confocal microscopy evaluation. Fifteen eyes of 14 patients (one bilateral case) showed Acanthamoeba keratitis alterations that ranged from massive infestation to cicatricial opacity in the stroma. Ten patients (71%) were females. Mean age was 26 years (range 19 to 37 yr). All patients were contact lens wearers. Confocal microscopy was a useful, noninvasive technique in the diagnosis and treatment of Acanthamoeba keratitis, especially in those cases in which corneal scraping, cytological analysis, and culture are negative. It also eliminated the necessity of tissue biopsy, considered an invasive procedure.