Pellicle is the initial proteinaceous layer that is formed almost instantaneously on all solid surfaces in the oral cavity. It is of essential relevance for any interactions and metabolism on the tooth surface. Up to now, there is no information on the metabolome of this structure. Accordingly, the present study aims to characterise the metabolomic profile of in-situ pellicle in children with different caries activity for the first time in comparison to saliva. Small molecules such as carbohydrates, amino acids, organic acids, and fatty acids, putatively involved in the formation of caries were quantified using mass spectrometry (MS)-based techniques, such as (stable isotope dilution analysis)-ultra-performance liquid chromatography-tandem MS and gas chromatography/electron ionisation-MS. Pellicle and corresponding saliva samples were collected from caries-active, caries-free and caries-rehabilitated 4- to 6-year-old children. The most abundant analytes in pellicle were acetic acid (1.2–10.5 nmol/cm2), propionic acid (0.1–8.5 nmol/cm2), glycine (0.7–3.5 nmol/cm2), serine (0.08–2.3 nmol/cm2), galactose (galactose + mannose; 0.035–0.078 nmol/cm2), lactose (0.002–0.086 nmol/cm2), glucose (0.018–0.953 nmol/cm2), palmitic acid (0.26–2.03 nmol/cm2), and stearic acid (0.34–1.81 nmol/cm2). Significant differences depending on caries activity were detected neither in saliva nor in the corresponding pellicle samples.