Significance Efforts to improve tuberculosis therapy include optimizing multidrug regimens to take advantage of drug–drug synergies. However, the complex host environment has a profound effect on bacterial metabolic state and drug activity, making predictions of optimal drug combinations difficult. In this study, we leverage a newly developed library of conditional knockdown Mycobacterium tuberculosis mutants in which genetic depletion of essential genes mimics the effect of drug therapy. This tractable system allowed us to assess the effect of growth condition on predicted drug–drug interactions. We found that these interactions can be differentially sensitive to the metabolic state, and select in vitro–defined interactions can be leveraged to accelerate bacterial killing during infection. These findings suggest strategies for optimizing tuberculosis therapy.