The cell wall of mycobacteria includes an unusual outer membrane of extremely low permeability. This cell envelope consists of a characteristic cell wall skeleton, a mycoloyl arabinogalactan peptidoglycan complex, and related hydrophobic components that contribute to the cell surface properties. In this study 1400-year-old mycolic acids as unique tuberculosis biomarkers have been extracted and identified for the first time by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) and Fourier transform infrared spectroscopy (FT-IR). The data suggest that the MALDI TOF MS has potential as a rapid and reproducible technique for the detection and identification of ancient mycobacterial infections.