Petroleum emulsion interfacial material (species that reside in the water/oil interface) are believed to be responsible for stabilizing emulsions in petroleum; therefore, their characterization can help to develop strategies to mitigate/manipulate petroleum emulsions. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has shown that the species present at the oil/water interface are enriched in sulfur- and oxygen-containing functionalities. However, structural and isomeric information about the chemical functionalities is still uncertain. Here, we demonstrate the potential of ion mobility mass spectrometry (IM-MS) combined with post-ion mobility collision-induced dissociation (post-IM CID) to characterize functional isomers in petroleum emulsion interfacial material. Interfacial material was isolated from Athabasca bitumen and a heavy crude oil by the wet silica method. IM time-of-flight (TOF) MS analysis shows the presence of multiple isomeric O3S1 species in both samples. Post-IM CID helps to identify the mobility-separated isomers; losses of CO2, H2O, and CO2 + H2O indicate the presence of a carboxylic group (naphthenic acids) and possibly a hydroxyl group, whereas losses of an alkyl chain to yield fragment ions with 4 double bond equivalents as well as the formation of SO3 suggest alkylbenzenesulfonates. Alkylbenzenesulfonates correspond to surfactants commonly added to petroleum, as confirmed by a comparison to a standard mixture of linear alkylbenzenesulfonates (LAS). The characterization and distinction of the isomers by CID is possible even in samples containing a mixture of functionalities by virtue of pre-separation by ion mobility. Emulsion interfacial material isolated from both Athabasca bitumen and heavy crude oil contains a mixture of functional isomers. Post-IM CID also allows for the use of diagnostic fragment ions to deconvolve incompletely resolved IM peaks. The deconvolution shows that synthetic surfactants (LAS) are more abundant in the emulsion interfacial material from the heavy crude oil, whereas naturally occurring SO3 naphthenic acids are predominant in Athabasca bitumen. FT-ICR mass spectral data confirm the IM-TOF molecular formula assignments and reveal that isobaric ions of different mobilities are indeed isomers.