Cholesterol plays critical functions in arranging the biophysical attributes of proteins and lipids in the plasma membrane. For various viruses, an association with cholesterol for virus entrance and/or morphogenesis has been demonstrated. Therefore, the lipid metabolic pathways and the combination of membranes could be targeted to selectively suppress the virus replication steps as a basis for antiviral treatment. U18666A is a cationic amphiphilic drug (CAD) that affects intracellular transport and cholesterol production. A robust tool for investigating lysosomal cholesterol transfer and Ebola virus infection is an androstenolone derived termed U18666A that suppresses three enzymes in the cholesterol biosynthesis mechanism. In addition, U18666A inhibited low-density lipoprotein (LDL)-induced downregulation of LDL receptor and triggered lysosomal aggregation of cholesterol. According to reports, U18666A inhibits the reproduction of baculoviruses, filoviruses, hepatitis, coronaviruses, pseudorabies, HIV, influenza, and flaviviruses, as well as chikungunya and flaviviruses. U18666A-treated viral infections may act as a novel in vitro model system to elucidate the cholesterol mechanism of several viral infections. In this article, we discuss the mechanism and function of U18666A as a potent tool for studying cholesterol mechanisms in various viral infections.