With the rapid rise in the prevalence of non-tuberculous mycobacteria (NTM) diseases across the world, the microbiological diagnosis of NTM isolates is becoming increasingly important for the diagnosis and treatment of NTM disease. In this study, the clinical presentation, species distribution and drug susceptibility of patients with NTM disease visiting the Chongqing Public Health Medical Centre during March 2016–April 2019 were retrospectively analysed. Among the 146 patients with NTM disease, eight NTM species (complex) were identified. The predominant NTM species in these patients were identified to be Mycobacterium abscessus complex (53, 36.3%), M. intracellulare (38, 26%) and M. fortuitum (17, 11.7%). In addition, two or more species were isolated from 7.5% of the patients. Pulmonary NTM disease (142, 97.3%) showed the highest prevalence among the patients. It was observed that 40.1% of the patients with pulmonary NTM disease had chronic pulmonary obstructive disease and bronchiectasis, while 22.5% had prior tuberculosis. Male patients showed more association with the conditions of cough and haemoptysis than the female patients. In an in vitro antimicrobial susceptibility testing, most of the species showed susceptibility to linezolid, amikacin and clarithromycin, while M. fortuitum exhibited low susceptibility to tobramycin. In conclusion, the prevalence of NTM disease, especially that of the pulmonary NTM disease, is common in Southwest China. Species identification and drug susceptibility testing are thus extremely important to ensure appropriate treatment regimens for patient care and management.