The acquired enamel pellicle (AEP) is a thin acellular film that forms on tooth surfaces upon exposure to the oral environment. It consists predominantly of salivary proteins but also includes non-salivary-derived proteins, carbohydrates, and lipids. AEP serves as an interface between teeth and the oral environment, playing a critical role in maintaining oral health by regulating processes including lubrication, demineralization, remineralization, and shaping the composition of early microbial flora adhering to tooth surfaces. Knowledge of the 3D structure of the AEP and how that correlates with its protective functions may provide insight into several oral pathological states, including caries, erosion, and periodontal disease. This chapter intends to inform readers about the latest discoveries related to the formation, ultrastructure, composition, and functions of the AEP and its close relationship with the formation of oral biofilm. Also, the authors will provide a perspective on the future of this area of investigation, with particular emphasis on the emerging role of proteomics and microscopy techniques in oral diagnosis and therapeutic application.