Background <p>Diagnosing pulmonary non-tuberculous mycobacterial (NTM) disease remains challenging, not only because its clinical manifestations often overlap with those of tuberculosis (TB), but also because its symptoms and signs—such as chronic cough, sputum production, fatigue, and weight loss—can closely resemble those of other common respiratory conditions, including chronic obstructive pulmonary disease (COPD), recurrent pulmonary infections, and chronic bronchitis. This symptomatic overlap often leads to delayed or misdiagnosis, a problem exacerbated by the time-consuming nature of confirmatory culture, which further impedes timely diagnosis.Against the backdrop of a rising incidence of NTM pulmonary disease, Nanopore Targeted Sequencing (NTS) emerges as a promising solution, enabling rapid and comprehensive pathogen detection. This study aims to evaluate the clinical utility of NTS, with a focus on its diagnostic performance, ability to delineate the pathogen spectrum, and reveal polymicrobial co-infections.</p> Methods <p>In this retrospective study, we analyzed 350 patients suspected with pulmonary NTM infection at Nanjing Second Hospital between 2023 and 2025. We evaluated clinical characteristics, pathogen profiles and co-infections. Bronchoalveolar lavage fluid (BALF), sputum, and tissue specimens were subjected to NTS. The diagnostic performance of NTS was assessed by comparing it with traditional methods, including acid-fast bacillus (AFB) smear, mycobacterial species identification, and conventional culture.</p> Results <p>Among 350 enrolled patients, 293 were diagnosed with NTM pulmonary disease. The NTM group was significantly older (median age: 61.0 vs. 43.5 years) compared to the non-NTM group (<i>n</i> = 57). NTM patients demonstrated significant cellular immune dysfunction, including lower CD3⁺, CD4⁺ T-cell levels, and CD4⁺/CD8⁺ ratio, alongside a more protracted disease course compared to controls. No significant difference in BMI was observed between the two groups.The most common clinical symptom was productive cough, and imaging predominantly showed patchy parenchymal and nodular shadows. Evaluation of diagnostic performance demonstrated that NTS showed the highest sensitivity (95.9%) and the highest AUC (0.892) among the methods evaluated, although its specificity (82.5%) was slightly lower than that of Mycobacterial culture (84.2%). In contrast, traditional methods such as Mycobacterial culture showed critically low sensitivity (50.5%), despite high specificity. Venn diagram analysis confirmed that NTS detected the highest number of positive cases, supporting its role as a comprehensive diagnostic tool. Furthermore, analysis of co-infections revealed a high frequency (56.0%) of polymicrobial infections among NTM patients, with <i>Mycobacterium tuberculosis</i> and Human Herpesvirus being the most common co-pathogens. Other frequent co-infections included <i>Aspergillus</i>, <i>Candida</i>, and <i>Pseudomonas aeruginosa</i>. Complex polymicrobial combinations were also observed, highlighting the intricate microbial landscape in NTM pulmonary disease and underscoring the need for comprehensive diagnostic strategies.</p> Conclusion <p>NTS is a rapid, highly sensitive tool that may improve the diagnosis of pulmonary NTM by enabling simultaneous species identification and co-infection profiling; however, its optimal application requires integration with clinical, radiological, and immunological findings to accurately distinguish true infection from colonization and guide effective management.</p>