Introduction: Acquired pellicle (AP) acts as a membrane preventing acids from coming into direct contact with the tooth. Possibly, individuals with different dental health status present changes in its composition that could disrupt this function. Thus, the aim of this study was to compare the protein composition of the AP in adolescents with erosive tooth wear (ETW), caries, or sound. Methods: Calibrated examiners in BEWE index and ICDAS-merged Epi criteria assessed ETW and caries in a sample of 454 systemically healthy adolescents aged 12–15 years old. Thirty subjects from that sample were selected for this study: ETW group (n = 10; total BEWE ≥9 and absence of dentinal caries lesions); caries group (n = 10; total BEWE <9 and with at least one dentinal caries lesion), and sound group (n = 10; total BEWE <9 and absence of dentinal caries lesions). Two-hour-formation AP samples were taken from buccal, occlusal/incisal, palatal/lingual tooth surfaces. Protein composition was analysed by liquid chromatography-tandem mass spectrometry. Using mean reporter ion values, relative abundances of proteins were compared among the three groups to calculate for fold changes. Twofold protein increases or decreases were reported (t test, p < 0.05). Gene Ontology (GO) of included proteins was assigned. Results: Mean age of participants was 13.1 ± 1.14 years and 56.6% were females. The prevalence of ETW was of 66.6% and of dentinal caries of 33.3%. The GO analyses showed that the majority of detected proteins were stress response related. The ETW group disclosed upregulated relative abundance of antileukoprotease (2.85-fold in ETW vs. sound and 2.34-fold in ETW group vs. caries group); histatin (2.42-fold in ETW group vs. sound group and 2.20-fold in ETW group vs. caries group), and prolactin-induced protein (2.30-fold in ETW group vs. sound group and 2.06-fold in ETW group vs. caries group) (p < 0.05). Hemoglobin subunits alpha (HBA) and beta (HBB) showed decreased relative abundances in the ETW and caries groups when compared to the sound group (HBA: 0.42-fold in ETW group and 0.40-fold in caries group; HBB: 0.45-fold in ETW group and 0.38-fold in caries group; p < 0.05). Conclusion: AP from individuals with ETW showed differences when compared to other dental conditions, with relative abundance increasing of some stress response-associated proteins in ETW and a decrease in proteins related to salivary protection against acid challenges.