We present results of the implementation of three-photon excitation fluorescence (3PEF) and third harmonic generation imaging measurements for the precise and nondestructive detection of natural and synthetic varnish layers, which are used for the surface protection of painted artifacts. For this purpose, we employ as an excitation source a compact femtosecond laser operating at 1028 nm. Two-dimensional images of the multilayer structures from different samples are depicted. The third harmonic signals show the interface between the different materials, when its refractive index mismatch is high enough. The depths of different layers of varnishes, presenting similar refractive index, are distinguishable with an axial resolution of approximately 1 microm by employing 3PEF measurements.