Some Brazilian crude oils form stable emulsions, even containing alow content of asphaltenes, recognized as the main natural surfactants in petroleum. This work aimed to understand the factors contributing to theformation and stabilization of these emulsions through the characterization of fractions with possible interfacial activity: interfacial materials (IM) and naphthenic acids (NA). For this, these fractions were extracted from three Brazilian crude oils with API between 21 and 30 degrees, allowing the characterization of their chemical composition and colloidal structure. Furthermore, these fractions and the residual oils from their extractions were used to prepare water-in-oil (W/O) emulsions. Chemical characterization carried out by elemental analysis, infrared spectroscopy, nuclear magnetic resonance, and mass spectrometry, showed that IM can be composed of saturates, aromatics, resins, and asphaltenes and NA by saturates and aromatics. However, they are enriched in aromatic compounds and polar groups, such as carboxylic acids, amines, sulfoxides,and sulfonic acids, concerning their original oils. Nevertheless, the NAfraction did not play a relevant role in emulsions. The amphiphilic compounds present in IM can form colloidal aggregates characterized by the small-angle X-ray scattering (SAXS) technique and promote the formation of emulsions. Still, they are not sufficient to guarantee their stabilization. The removal of IM increased the self-organization of aggregates in the residual crude oil and the stability of emulsions, which seems to be more related to rheological properties than to the interfacial activity of the compounds.