It is a growing problem around the world to deal with nontuberculous mycobacteria infection (NTM), but its clinical significance is still largely unknown. This study aims to investigate the epidemiology of NTM infections from various clinical samples and determine their clinical significance. From December 2020 to December 2021, 6125 clinical samples were collected. In addition to phenotypic detection, genotypic detection through multilocus sequence typing (hsp65,rpoB, and16S rDNAgenes) and sequencing was also conducted. Records of patients were consulted for clinical information, such as symptoms and radiological findings. Of the 6,125 patients, 351 (5.7%) were positive for acid-fast bacteria (AFB). Out of 351 AFB, 289 (82.3%) and 62 (17.7%) subjects were identified asM. tuberculosiscomplex (MTC) and NTM strains, respectively. Isolates ofMycobacterium simiaeandM. fortuitumwere the most frequent, followed by isolates ofM. kansasiiandM. marinum. We also isolatedM. chelonae,M. canariasense, andM. jacuzzii, which are rarely reported. Symptoms (P= 0.048), radiographic findings (P= 0.013), and gender (P= 0.039) were associated with NTM isolates.M. Fortuitum,M. simiae, andM. kansasiipresented with bronchiectasis, infiltration, and cavitary lesions most frequently, while cough was the most common symptom. In conclusion,Mycobacterium simiaeandM. fortuitumwere presented in seventeen and twelve NTM isolates from the collected samples. There is evidence that NTM infections in endemic settings may contribute to the dissemination of various diseases and the control of tuberculosis. In spite of this, further research is needed to evaluate the clinical significance of NTM isolates.