Aim: This study aimed to assess the role of environmental microbiota in the transmission of drug-resistant tuberculosis (DR-TB) by comparing microbiota composition, environmental exposures, and drug resistance patterns in individuals with DR-TB and healthy controls. Materials and Methods: A longitudinal cohort design was employed with 120 participants, including 60 individuals diagnosed with DR-TB (both MDR-TB and XDR-TB) and 60 healthy controls. Participants were recruited from a tertiary care hospital, and environmental samples (air, dust, and surface swabs) were collected every three months. Sputum samples were obtained at baseline and monthly intervals. High-throughput sequencing (16S rRNA gene sequencing) was used to analyze microbiota diversity, and drug resistance testing was performed using phenotypic and genotypic methods. Results: The DR-TB group exhibited significantly higher levels of environmental exposure to poor ventilation (63.33% vs. 36.67%), unsanitary conditions (56.67% vs. 30.00%), and shared living spaces (75.00% vs. 61.67%) compared to the control group. Microbiota analysis showed lower alpha diversity in the DR-TB group (3.12 ± 0.58 vs. 3.56 ± 0.52, p = 0.005). Drug resistance patterns revealed high resistance to Rifampin (90.00%), Isoniazid (93.33%), and Fluoroquinolones (60.00%) in the DR-TB group, with no resistance observed in the control group. Conclusion: This study highlights the significant role of environmental exposures and microbiota composition in the transmission of drug-resistant tuberculosis. The DR-TB group was more likely to be exposed to poor environmental conditions, which correlated with altered microbiota diversity and increased drug resistance. These findings underscore the importance of addressing environmental factors and the microbiome in TB control strategies.