Objectives: Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is widely used for identification of microorganisms in clinical diagnostics. However, some Mycobacterium species show low-quality spectra with standard preparation methods. This shortcoming was resolved using an optimized preparation technique. Mycobacterium spp. strains from interlaboratory tests between 2000 and 2012 (n = 22) and clinical isolates (n = 20) were analysed to evaluate the performance of the new preparation method and a corresponding developed database. Methods: Strains were cultivated in a BD BACTEC(TM) MGIT(TM) 320 system or on Löwenstein-Jensen medium (BD, Heidelberg), respectively. Samples were processed using a zirconia/silica bead based method and mass spectra were recorded with a microflex LT. The acquired mass spectra were compared to Mycobacteria Library 2.0 reference spectra using MALDI Biotyper 3.1 software (Bruker Daltonik, Germany). Results: MALDI Biotyper 3.1 and Mycobacteria Library 2.0 resulted in 41 (97.6%) identifications with log(score) values ≥ 2.0 corresponding to an identification at species level. This was achieved for the following species: M. abscessus, M. avium, M. bohemicum, M. celatum, M. chelonae, M. fortuitum, M. gordonae, M. interjectum, M. intracellulare, M. kansasii, M. malmoense, M. marinum, M. nebraskense, M. parascrofulaceum, M. phlei, M. scrofulaceum, M. shimodei, M. simiae, M. smegmatis, M. szulgai, and M. xenopi. Mass spectra of one strain (2.4%) yielded a log(score) value of 1.83. This isolate was assigned to M. scrofulaceum by MALDI Biotyper analysis and to M. paraffinicum/seoulense/scrofulaceum by 16S rRNA gene sequencing which cannot distinguish between these closely related species. Conclusion: A bead beating-based mycobacterial protein extraction in combination with Mycobacteria Library 2.0 was successfully applied for all interlaboratory test strains (n = 22) and almost all clinical isolates (19 out of 20). These data indicate a high potential of MALDI-TOF MS for fast and reliable identification of nontuberculous mycobacteria.