When mycobacteria are recovered in clinical specimens, timely species-level identification is required to establish the clinical significance of the isolate and facilitate optimization of antimicrobial therapy. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been reported to be a reliable and expedited method for identification of mycobacteria, although various specimen preparation techniques and databases for analysis are reported across studies. Here we compared two MALDI-TOF MS instrumentation platforms and three databases: Bruker Biotyper Real Time Classification 3.1 (Biotyper), Vitek MS Plus Saramis Premium (Saramis), and Vitek MS v3.0. We evaluated two sample preparation techniques and demonstrate that extraction methods are not interchangeable across different platforms or databases. Once testing parameters were established, a panel of 157 mycobacterial isolates (including 16 Mycobacterium tuberculosis isolates) was evaluated, demonstrating that with the appropriate specimen preparation, all three methods provide reliable identification for most species. Using a score cutoff value of ≥1.8, the Biotyper correctly identified 133 (84.7%) isolates with no misidentifications. Using a confidence value of ≥90%, Saramis correctly identified 134 (85.4%) isolates with one misidentification and Vitek MS v3.0 correctly identified 140 (89.2%) isolates with one misidentification. The levels of accuracy were not significantly different across the three platforms (P = 0.14). In addition, we show that Vitek MS v3.0 requires modestly fewer repeat analyses than the Biotyper and Saramis methods (P = 0.04), which may have implications for laboratory workflow.