Nontuberculous mycobacteria (NTM) are easily misdiagnosed as multidrug-resistant tuberculosis (MDR-TB), and treatment drugs are very limited. The main objective of our study was to evaluate the minimal inhibitory concentration (MIC) in vitro of bedaquiline (BDQ), clofazimine (CFZ), linezolid (LZD), delamanid (DLM), and pretomanid (PA-824) for treatment of and . Furthermore, we determined whether , and were related to drug resistance to provide an experimental basis for the use of these five drugs in the treatment of NTM.We identified sample characteristics of epidemics in 550 patients with suspected NTM infection in Nanjing from 2019 to 2021 using the PCR-reverse spot hybrid method. Furthermore, we evaluated the MIC of BDQ, CFZ, DLM, LZD, and PA-824 against 155 clinical isolates of NTM using the microbroth dilution method. The resistant isolates were sequenced using Sanger sequencing.The top three dominant species of NTM distributed in Nanjing were , and . Notably, the proportion of infections increased. The proportion of increased from 12% in 2019 to 18% in 2021. Demographic analysis showed that female infection rates were substantialy greater than male for (P=0.017, <0.05). Our results demonstrate that NTM are highly sensitive to bedaquiline and clofazimine in vitro. However, delamanid and pretomanid had little effect on and . In addition, we found 30-41 nucleotide deletion mutations and some novel point mutations in the gene of that are resistant to clofazimine.Bedaquiline, clofazimine, and linezolid were more successful in vitro treatments against and . The mutation may be associated with resistance of to clofazimine.