Although tuberculosis burden is generally declining, the relative importance of nontuberculous mycobacteria (NTM) is increasing. In South Korea, few studies have evaluated Mycobacterium tuberculosis complex (MTBC) and NTM detection patterns and species distribution using long-term data. We retrospectively compared MTBC and NTM detection patterns using respiratory specimen-based laboratory data from a tertiary care hospital in South Korea (2016–2025). We analyzed 48,533 specimens, and the sum of annual patient counts after within-year deduplication was 30,275. Patient-level positivity decreased for MTBC (4.7% to 2.4%) but increased for NTM (7.7% to 10.3%), with corresponding changes at the specimen level. Age- and sex-adjusted annual odds ratios (95% confidence intervals) were 0.913 (0.892–0.934) for MTBC and 1.009 (0.994–1.023) for NTM; the overall linear NTM trend was nonsignificant. NTM-positive patients were older than MTBC-positive patients (median, 73 vs. 64 years). Co-detection of MTBC–NTM or multiple NTM species or subspecies was also observed. MTBC positivity declined, whereas NTM positivity increased mainly during 2016–2019; the relative proportion of NTM detections increased. Co-detection in the same specimen suggests the importance of differentiating MTBC from NTM and identifying NTM. Our findings may inform the development of institutional laboratory diagnostic strategies.