The study investigated the factors influencing the turnover time (TAT) in the evaluation of cultures of non-tuberculous mycobacteria (NTM) in a tertiary care hospital from June 2022 to May 2024. 289 NTM strains were included, of which 22 were fast-growing (RGM) with a median TAT of 7 days and 267 were slow-growing (SGM) with a median TAT of 11 days, a statistically significant difference (p < 0.001). Different species within RGM and SGM showed significant differences in TAT. Patients with NTM lung disease, bronchiectasis, or cavitation had a shorter median TAT (8–9 days) compared to patients without these findings (p < 0.05). TAT was negatively correlated with the degree of acid-fast bacilli smear (correlation coefficient -0.490, p < 0.001). Acid-fast bacilli smear result was the only independent factor affecting TAT (Wald χ² = 39.71, p < 0.001). RGMs had a shorter TAT than SGMs, and TAT was associated with clinical diagnoses and imaging findings.