Interfacial chemistry and physics dominate fluid distribution within crude oil reservoirs and the efficiency of hydrocarbon recovery. For improvements to be made in extraction and production processes, it is essential that the complex boundaries between the different reservoir phases are better understood. The purpose of the present paper is to describe a multi-technique study of the spreading behavior and composition of so-called precursor films deriving from natural bitumen extracted from Athabasca oil sands at an air-water interface. Film spreading on deionized water and saline subphases was followed using ellipsometry and film pressure measurements. The films comprise surface-active bitumen components which could potentially influence the surface chemistry of the reservoir, with implications for mineral wettability, hydrocarbon recovery and the formation and stabilization of oil/water emulsions and oil foams during production. Infrared spectroscopy and mass spectrometry identified the presence of carbonyl groups as well as various classes of sulfur and oxygen hetero-species in the precursor films.