The purpose of this study was to investigate oral microbiome and host proteins in archaeological human dental tissues using a shotgun proteomics approach. The research focuses on dental calculus (mineralized plaque), dentine, a carious lesion, and an alveolar bone abscess from the medieval site of Dalheim, Germany (ca. AD 950-1200). For comparison, proteins were also analyzed from archaeological faunal dental tissues and human dental calculus samples from modern Swiss dental patient controls. Protein extraction and generation of tryptic peptides from tooth and dental calculus specimens was performed using a filter-aided sample preparation (FASP) protocol, modified for mineralized and degraded samples. Total protein extraction was performed on a total of fourteen samples: four ancient human calculus samples (indicated as: G12, B71, B61, and B78), four ancient human tooth root samples (indicated as: G12, B17, B61, and B78), one carious lesion (indicated as: B17), one alveolar bone abscess (indicated as: B17), two ancient fauna crown cementum/calculus samples (indicated as: F1 [sheep] and F5 [cattle]), and two modern dental calculus samples from clinical patients (indicated as: P1 and P2). All samples were extracted at the Centre for Evolutionary Medicine (ZEM) at the University of Zürich with the exception of dental calculus from G12, P1, and P2, which were extracted at the Center for GeoGenetics (CGG) at the University of Copenhagen. Two samples (G12 and B61 calculus) were extracted a second time in an independent laboratory at the University of York (YORK) for comparison. Sample extracts were then sequenced (LC-MS/MS) at the Functional Genomics Center Zürich (FGCZ) using an LTQ-Orbitrap Velos, at the Novo Nordisk Foundation Center for Protein Research (CPR) using a Q-Exactive Hybrid Quadrupole Orbitrap, and at the University of York’s Proteomics and Analytical Biochemistry Laboratories (PABL) using a MaXis UHR-Qq-TOF.