The formation of stable emulsions between produced water and crude oil represents, in part, one of the challenges of the crude oil production and exploitation industry. The problems related to stable oil emulsions affect both Upstream and Downstream processes. In this sense, the aim of this work was to evaluate, at the molecular level, the species that exhibit interfacial activity within these systems. For this purpose, 5 crude oils of different ⁰API were selected, these were named OB-1 (⁰API 23.5), OB-2 (⁰API 7.4), OB-3 (⁰API 11.0), OB-4 (⁰API 15.4) and OB-5 (⁰API 22.4). The parameters: stability time and Zeta Potential of the system were used for correlation with the chemical nature of the active species present in the Interfacial Materials (IMs). The molecular-level characterization of the IMs extracted from the oils studied was performed using an ultra-high-resolution mass spectrometer Orbitrap type, adopting APPI as the ionization source. Regarding the results of the stability test, it was observed that the OB-2 oil was the one that kept the system homogeneous for the longest time. About the zeta potential of the systems, the following values were observed: -17.1 mV, -12.2 mV, +16.5 mV, +23.7 mV, -44.3 mV, for the oils OB-1, OB-2, OB-3, OB-4 and OB-5, respectively. According to the XDLVO theory for a non-aqueous system, the closer to zero the absolute value of the zeta potential is, the greater the stability of the system formed. Thus, the zeta results corroborated the observed stability times. Considering these results, and correlating them with the mass content of the isolated interfacial materials, no linear relationships were observed, therefore, the number of active species is not the determining factor for the stability of the emulsion system, but, rather, its nature. Thus, the IMs were fractionated and characterized by APPI(+)-FT-Orbitrap MS. The majority classes were: OxSy (x = 1 or 3, and y = 1 or 2) and Nx and NxOy (x = 1 and y = 1 or 2). By performing linearization of the zeta potential with the majority classes obtained by APPI(+)-FT-Orbitrap MS it was observed that the polar (acidic) species belonging to the OxSy classes (the most abundant species) act as natural surfactants, because, they showed positive Pearson correlation coefficients, between 0.55 and 0.64, indicating moderate to high correlation. The O3S1 and O2S2 classes are associated with sulfur-containing naphthenic acids and are described as natural surfactants. On the other hand, the Nx and NxOy species act in the opposite way, that is, they provide a greater disturbance of the system because, from the negative Pearson's relationship, it was observed that the higher the intensity of the N1 and N1O1 classes, the lower the stability of the resulting system. Therefore, for the set of oils studied, oils that have acidic molecular structures composed of oxygen-sulfur-containing compounds confer greater stability to the emulsion system involving crude oils. And conversely, the greater the presence of nitrogen-containing compounds, and nitrogen-oxygen-containing compounds, the lower the stability of the resulting system.