Nontuberculous mycobacteria (NTM) and Nocardia are often isolated via mycobacterial culture, yet their prevalence in various pulmonary specimens is uncertain. We retrieved mycobacterial culture results from pulmonary specimens from a tertiary hospital in China between January 2017 and July 2021. The prevalence of non-Mycobacterium tuberculosis (non-Mtb, including NTM and Nocardia) has surpassed Mtb, accounting for 1.7 times the rate of Mtb among all isolates. The frequency of non-Mtb positivity in sputum specimens was significantly higher compared to that in bronchoscopy specimens (3.7% vs 1.9%, P <
0.001). Furthermore, NTM accounted for 59.8% of all Mycobacterium isolates and 42.1% of patients with positive acid-fast staining. Multivariate logistic regression analysis identified several risk factors significantly associated with non-Mtb isolates compared to Mtb, including age over 45 years (adjusted odds ratio [aOR] 2.27), bronchiectasis (aOR 4.52), pulmonary interstitial disorder (aOR 2.11), chronic obstructive pulmonary disease (aOR 1.94), and non-diabetes (aOR 2.10).