The asphaltene fraction of crude oil is commonly considered to be responsible for the formation of highly undesirable, stable water-in-crude oil emulsions and rag layers. We developed a new procedure for subfractionation of asphaltenes based on their interfacial activity. The most interfacially active asphaltene (IAA) subfraction was extracted as an interfacial material from emulsified water droplets in asphaltene solution. The IAA subfraction represents less than 2 wt % of whole asphaltenes (WA), but its removal had a profound effect on interfacial and thin emulsion film properties. It was found that the IAA subfraction is a main contributor to emulsion stabilization and formation of rigid oil-water interfaces. IAA adsorbed irreversibly at the oil-water interface and formed interfacial layers of low compressibility. Thin emulsion films of toluene stabilized by IAA were thicker and exhibited severe aging effects in contrast to the films stabilized with remaining asphaltene fractions, which were thin and less rigid, without any noticeable aging effects.