Background: Respiratory specimens subjected to mycobacterial detection were initially pre-treated with N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) to remove the mucus and normal flora. Next, they were washed and neutralized with phosphate-buffered solution (PBS). The effectiveness of distilled water (DW) compared to PBS as a washing neutralizer during identification of mycobacteria was evaluated in this study. Methods: We analyzed the results of mycobacterial test conducted at a general hospital in Gwangju from October 2016 to September 2018. PBS and DW were used as a respiratory sample washing agent for one year each. Results: The positive culture rate for the culture of mycobacteria was 12.7% (1,843/14,532) and 14.7% (2,095/14,291), when PBS and DW were used, respectively. The recovery rate of the mycobacteria growth indicator tubes (MGIT) and the separation rates of Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM) showed no significant change. However, in 2% Ogawa medium, as the NTM culture increased from 47.4% (399/841) to 56.1% (630/1,122), the recovery rate increased from 45.6% (841/1,843) to 53.6% (1,122/2,095). The MGIT contamination rate decreased from 6.5% to 4.1%. Conclusion: DW as a washing agent for NALC-NaOH increased the recovery rate of Ogawa media and reduced the contamination rate of MGIT. Therefore, use of DW instead of PBS as a washing neutralizer during identification of mycobacteria might be useful.