Objective To investigate the value of metagenomic next-generation sequencing (mNGS) in the rapid diagnosis of clinical mycobacterial infections. Methods The clinical data of 782 hospitalized patients with suspected mycobacterial infections from April 2023 to December 2025 were retrospectively analyzed. The diagnostic performance of mNGS was compared with that of conventional methods, including culture and acid-fast staining. Results Among the 782 patients, 465 were ultimately diagnosed with mycobacterial infections, including 388 cases of Mycobacterium tuberculosis infection and 77 cases of non-tuberculous mycobacterial infection. Using the final clinical diagnosis as the gold standard, the sensitivity, specificity, positive predictive value, and negative predictive value of mNGS for diagnosing mycobacterial infections in all specimens were 52.5%, 79.8%, 79.2%, and 53.4%, respectively; those of culture were 1.9%, 100.0%, 100.0%, and 41.0%, respectively; and those of acid-fast staining were 14.2%, 99.6%, 98.4%, and 41.1%, respectively. The areas under the receiver operating characteristic curves for mNGS, culture, and acid-fast staining in evaluating the diagnosis of mycobacterial infections were 0.66 [95% confidence interval (CI) (0.63, 0.69)], 0.51 [95%CI (0.50, 0.52)], and 0.57 [95%CI (0.55, 0.59)], respectively. Conclusion mNGS demonstrates certain sensitivity, specificity, and negative predictive value in the diagnosis of mycobacterial infections, and may serve as a reference for early clinical diagnosis.