Resistance of Acanthamoeba castellanii cysts to disinfection agents, antimicrobial agents, heat, freeze–thawing, ultraviolet radiation (UV), γ irradiation, and cellulase were evaluated in vitro. Following exposure to different agents, the cysts were removed and cultured for A. castellanii trophozoites for 3–14 days. Solutions containing 20% isopropyl alcohol or 10% formalin effectively killed A. castellanii cysts. Hydrogen peroxide (3%, AOSept Disinfectant) effectively killed A. castellanii cysts after 4 hr of exposure. Polyhexamethylene biguanide (0.02%), clotrimazole (0.1%), or propamidine isethionate (Brolene) were effective in killing A. castellanii cysts in vitro. Acanthamoeba castellanii cysts were resistant to both 250 K rads of γ irradiation and 800 mJ/cm² of UV irradiation. Excystment of trophozoites was accelerated after exposure to 10, 100, and, 1,000 units of cellulase. These results suggest that A. castellanii cysts benefit by enhanced survival because of their resistance to very harsh environmental conditions.