to achieve a comprehensive understanding of the characteristics of patients with non-tuberculous mycobacteria (NTM), patients with NTM between January 2016 and June 2019 were recruited from a primary hospital. NTM were identified based on the MBP64 protein assay. The clinical records and laboratory assay results were retrospectively reviewed. A total of 204 patients with NTM were included in the final analysis. The patients with multiple isolations were more likely accompanied with chronic obstructive pulmonary disease (copD) (p = 0.029) and arthritis (p = 0.049), but showed a lower percentage of positive t-spot results (p = 0.022). In addition, patients with multiple isolations showed a higher rate of positive acid-fast staining results and their symptom duration was more likely longer than 30 days (p = 0.019). Patients with a positive response in T-spot assay showed a higher proportion of nodular manifestation on computed tomography (ct) than those with a negative response. compared with male patients with ntM, female patients showed lower rates of positive acid-fast staining results (p = 0.03), but were more likely accompanied with COPD (p < 0.0001). The positive acid-fast staining results were closely associated with pulmonary cavities and tuberculosis antibody. patients with different NTM isolation frequencies were closely associated with coexisting diseases and examination results.