The anti-interferon-gamma (IFN-gamma) autoantibody is a known cause of opportunistic non-tuberculous mycobacterial (NTM) infection in adults. Diagnosis of those patients is difficult due to the low sensitivity of bacterial culture, and because detection of the neutralizing autoantibody needs special laboratory devices. We conducted a retrospective review of indirect and inhibitory ELISA, both used for detection of anti-IFN-gamma auto-antibody in 102 patients with lymphadenopathies. We assessed hospital records of NTM isolation and/or diagnosis of NTM infection. The review revealed the compatible sensitivity and superior specificity and predictive values for inhibitory ELISA over against indirect ELISA-the latter achieving 100% specificity and positive predictive value for diagnosis of NTM infection in patients with lymphadenopathies. The results confirm functional assays that show plasma samples from NTM-infected patients with positive results by either indirect and/or inhibitory ELISA are IFN-gamma neutralizing autoantibodies. The inhibitory titer of anti-IFN-gamma auto-antibody can be used to distinguish patients with active from inactive NTM infection. Inhibitory ELISA is thus a practical, rapid, high performance tool for routine detection of anti-IFN-gamma autoantibody and NTM infection diagnosis before confirmation, enabling a timely therapeutic strategy for active infection treatment.