The film structure of a nonlinear optical azo dye (Disperse Red-1, DR-1) was probed by a combination of spectroscopic and microscopic techniques. Second harmonic generation (SHG) and linear dichroism provided evidence for the spontaneous formation of DR-1 multilayers at a 2-propanol/fused-silica interface under equilibrium conditions. Specifically, the adsorption isotherm measured by SHG exhibited two distinct adsorption regimes; the second regime (>10-3 M) being indicative of multilayer formation in which some degree of polar order was retained. The trend in apparent tilt angle measured by linear dichroism was consistent with multilayer formation at solution-phase concentrations above 10-3 M. Multilayer formation was directly observed, on a dip-coated substrate, by atomic force microscopy and further confirmed by ellipsometry. The significance of spontaneous multilayer formation with z-axis polar order in higher layers is the potential for growth of molecular multilayer systems without the need for covalent or sequential deposition.