Successful tuberculosis (TB) treatment requires at least six months of combination therapy. Even with this prolonged treatment, failure and relapse still occur in 5–10% of cases and are often linked to the emergence of multidrug resistance. Therefore, a deeper understanding of bacterial determinants of antibiotic efficacy is essential to uncover novel mechanisms of antibiotic resistance and to facilitate the development of more effective combinatorial TB therapies. In this work, I combined bacterial functional genetics and genomics to investigate the mechanisms influencing antibiotic efficacy in Mycobacterium tuberculosis (Mtb). First, I leveraged a library of conditional knockdown mutants to characterize Mtb chemical-genetic interactions (CGIs) in mouse TB infection and treatment models. This work uncovered a range of synergistic and antagonistic CGIs specific to the infection environment, revealing novel targets for combinatorial TB therapeutics. Specifically, we identified previously unrecognized determinants of Mtb susceptibility to pyrazinamide (PZA), showing increased PZA efficacy in thiamine and purine metabolism mutants, and reduced activity in mutants of the ESX-3 type VII secretion system. Second, using a combination of genomic analysis and in vitro experimentation, I identified simple sequence repeat (SSR) indels associated with decreased antibiotic susceptibility during TB infection. Notably, we identified triplet SSR indels in ppe53 and ponA1 that are enriched in drug-resistant TB and confer intermediate in vitro multidrug resistance. Together, this work defines infection-specific mechanisms of antibiotic activity, identifies potential targets for combinatorial TB drug development, and reveals a previously unrecognized role for triplet SSRs as determinants of intermediate antibiotic resistance in Mtb.