Drug-resistant tuberculosis (DR-TB) is still a concern for public health and health security, making TB the world’s largest cause of death from a single infectious agent. This Chapter was aimed at evaluating the prevalence of DR-MTB in rural communities of South Africa. A cross-sectional study in rural Vhembe District, South Africa, recruited 175 active TB patients. Data on lifestyle behaviour, socioeconomic, and environmental characteristics were collected via a questionnaire, and samples (blood and sputum) were collected. The U-rapid blood test confirmed HIV status. DNA was extracted from sputum and tested using multiplex real-time PCR assays with Anyplex and Allplex kits to detect Mycobacterium tuberculosis/nontuberculous mycobacteria, as well as DR-TB strains, respectively. The participants’ ages ranged from 18 to 82 years (mean = 44 ± 13.0), with the majority being males (57.1%, 100/175) and unemployed (61.1%, 107/175). The co-infections detected include NTM and HIV. The prevalence of DR-MTB was 2.3% (4/175). A 20.6% (36/175) success rate was achieved in treatment outcomes. DR-TB remains low and is effectively managed, with favourable treatment outcomes reflecting effective clinical and public health interventions. However, the high prevalence of emerging NTM and HIV infections may compromise these gains, posing a potential threat to sustained TB control.