Petroleum molecules with interfacial activity play a fundamental role in stabilizing water-in-oil (W/O) emulsions and thus have been the focus of many studies. Using the wet silica method, crude oil samples from Brazilian fields (designated PS and P6) were fractionated into two fractions-one with the interfacially active molecules (IA) and another containing the noninterfacially active molecules (NIA). This fractionation was performed using a mixture of cyclohexane/toluene (1:1 v/v) (CH/TOL) for oils PS and P6 and n-octane/toluene (1:1 v/v) (OC/TOL) only for oil P6 to evaluate the influence of the solvent system on the profile of the fractions obtained. The fractions obtained and the parent oils were characterized chemically by Fourier-transform infrared spectroscopy (FTIR), H-1-nuclear magnetic resonance (H-1-NMR), and electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry (ESI (+/-) FT-ICR MS) and analyzed for interfacial activity using systems composed of brine (aqueous phase) and n-heptane/toluene mixture (organic phase). The results from FTIR and H-1-NMR evidenced the polar and aromatic character of the IA fractions, while the predominantly hydrocarbon composition was observed in the NIA fractions as well as their respective parent oils. The characterization by FT-ICR MS showed the influence of the solvent system on the basic species of the IA fractions, by ESI (+), and a difference in the acid compounds of the fractions isolated from the two parent oils, by ESI (-): the IA fraction isolated from PS presented the compound classes O-4[H] and O-2[H] as major components, while the major compounds in the IA fractions isolated from P6 with CH/TOL or OC/TOL were O[H], O-2[H], and N[H]. Decrease in the interfacial tension values on the order of 30% was observed when comparing each interfacially active fraction with their parent oil.