Mycobacterium tuberculosis (Mtb) is evolutionarily equipped to resist exogenous reactive oxygen species but shows vulnerability to an increase in endogenous ROS (eROS). Since eROS is an unavoidable consequence of aerobic metabolism, understanding how eROS levels are controlled is essential yet remains uncharacterized. By combining the Mrx1-roGFP2 redox biosensor with transposon mutagenesis, we identified 368 genes (redoxosome) responsible for maintaining non-toxic levels of eROS in Mtb. Integrating redoxosome with a global network of protein-protein interactions and transcriptional regulators revealed a hypothetical protein (rv0158) as a top node managing eROS and redox homeostasis in Mtb. RNA sequencing, seahorse XF flux measurements, and lipid analysis indicate that rv0158 is required to balance the deployment of fatty acid substrates between lipid anabolism and oxidation. Disruption of rv0158 perturbed redox balance in a carbon-source-specific manner, promoted killing in response to anti-TB drugs, reduced survival in macrophages, and lowered persistence in mice.