Acanthamoeba species are an important pathogen that causes Acanthamoeba keratitis, which causes a visual loss, and encephalitis, which lead to the central nervous system infection and death. The options available to successfully treat Acanthamoeba infections have limited success. New therapeutic approaches must thus be developed, and especially combination medication therapy may be a successful and an effective strategy. The aim of this study was to assess the combination efficacy of verapamil and fluconazole against Acanthamoeba trophozoites and cysts. The effects of drugs on growth inhibition against Acanthamoeba were tested using amoebicidal assays. The viability of Acanthamoeba was assessed using Trypan blue and hemocytometer counts. The effect of three different concentrations of fluconazole, verapamil and fluconazole + verapamil combination on growth inhibition against Acanthamoeba trophozoites and cysts was significant compared to the control (p