Cholesterol plays a crucial role in modulating the physicochemical properties of biomembranes, both increasing mechanical strength and decreasing permeability. Cholesterol is also a common component of vesicle-based delivery systems, including liposome-based drug delivery systems (LDSs). However, its effect on the partitioning of drug molecules to lipid membranes is very poorly recognized. Herein, we performed a combined experimental/computational study of the potential for the use of the LDS formulation for the delivery of the antifungal drug itraconazole (ITZ). We consider the addition of cholesterol to the lipid membrane. Since ITZ is only weakly soluble in water, its bioavailability is limited. Use of an LDS has thus been proposed. We studied lipid membranes composed of cholesterol, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), and ITZ using a combination of computational molecular dynamics (MD) simulations of lipid bilayers and Brewster angle microscopy (BAM) experiments of monolayers. Both experimental and computational results show separation of cholesterol and ITZ. Cholesterol has a strong preference to orient parallel to the bilayer normal. However, ITZ, a long and relatively rigid molecule with weakly hydrophilic groups along the backbone, predominantly locates below the interface between the hydrocarbon chain region and the polar region of the membrane, with its backbone oriented parallel to the membrane surface; the orthogonal orientation in the membrane could be the cause of the observed separation. In addition, fluorescence measurements demonstrated that the affinity of ITZ for the lipid membrane is decreased by the presence of cholesterol, which is thus probably not a suitable formulation component of an LDS designed for ITZ delivery.