The interfacial behavior of asphaltene samples, obtained from two different sources, was studied by means of Langmuir balance and Brewster angle microscopy (BAM) at the air/water interface. Transferred films from the air/water interface on mica were studied by atomic force microscopy (AFM). Isotherms obtained from the Langmuir balance experiments for all samples are qualitatively similar and indicate that their interfacial behavior may be correlated to the molecular weight of the asphaltene samples. At mesoscopic scales, BAM images of asphaltene films deposited on the water surface reveal that asphaltene samples do not form a monomolecular film, as proposed in the literature, but rather a mixture of 2D and 3D domains. This behavior is confirmed when, on a hydrophilic surface such as mica, the film is transferred from the Langmuir trough and is found to be not homogeneous or monomolecular. AFM images revealed the formation of an irregular and meandering layer of 10.2-12.5 nm thickness. Initially, the films exhibit some mesostructures graphically identified as small and large disklike structures; these disks coalesce or fuse into more complex mesostructures: drumsticklike and ribbonlike structures. A closer look revealed that these mesostructures are formed by small colloidal-like particles or small micelles (< 100 nm). The spreading capacity of asphaltene on the mica surface seems to be related to the separation methods and the amount of resins associated with the asphaltenes. The complete coverage of the surface and the formation of an irregular 3D film occur as a function of aging time or when the transference pressure of the asphaltene film from water to mica is increased.