Over the past few decades, there has been a significant increase in the number of mycobacterial species described. Currently, the genus Mycobacterium consists of 170 species. Most species are called nontuberculous mycobacteria (NTM) and are potentially or rarely pathogenic and ubiquitous. One of the main challenges in mycobacteriology is the rapid and precise identification of these microorganisms. In this work, we compared two protein extraction protocols for the identification of 38 reference strains and clinical isolates, representing 27 species, by mass spectrometry (MALDI-TOF MS) to subsequently use the best method for identifying environmental mycobacteria. The results obtained with reference strains and clinical isolates showed that protocol A was effective in identifying 92.1% of mycobacterial specimens at the species level and protocol B, 50%. Therefore, protocol A was evaluated for the rapid identification of 27 environmental mycobacterial isolates. These isolates were subjected to PCR-restriction enzyme analysis (PRA-hsp65). Two isolates were misidentified by PRA-hsp65, whereas MALDI-TOF MS was able to identify them correctly. The results were confirmed by hsp65 and 16S rRNA gene sequencing. Mass spectrometry has the advantage of being a simpler and faster technique than PRA-hsp65, and our results showed that MALDI-TOF MS is a valuable tool for the identification of environmental mycobacterial isolates.