The study investigates the role of mitochondrial reactive oxygen species (mROS) in macrophage bactericidal activity, particularly in response to Toll-like receptor (TLR) signaling. The authors demonstrate that engagement of TLR1, TLR2, and TLR4 leads to the recruitment of mitochondria to macrophage phagosomes and enhances mROS production. This process involves the translocation of tumor necrosis factor receptor-associated factor 6 (TRAF6) to mitochondria, where it interacts with evolutionarily conserved signaling intermediate in Toll pathways (ECSIT). The interaction between TRAF6 and ECSIT results in ECSIT ubiquitination and enrichment at the mitochondrial periphery, leading to increased mROS generation. Depletion of ECSIT or TRAF6 in macrophages results in reduced mROS production and impaired ability to kill intracellular bacteria. Additionally, reducing mROS by expressing catalase in mitochondria also impairs bacterial killing. These findings reveal a novel pathway linking innate immune signaling to mitochondria and highlight the importance of mROS in antibacterial responses.The study investigates the role of mitochondrial reactive oxygen species (mROS) in macrophage bactericidal activity, particularly in response to Toll-like receptor (TLR) signaling. The authors demonstrate that engagement of TLR1, TLR2, and TLR4 leads to the recruitment of mitochondria to macrophage phagosomes and enhances mROS production. This process involves the translocation of tumor necrosis factor receptor-associated factor 6 (TRAF6) to mitochondria, where it interacts with evolutionarily conserved signaling intermediate in Toll pathways (ECSIT). The interaction between TRAF6 and ECSIT results in ECSIT ubiquitination and enrichment at the mitochondrial periphery, leading to increased mROS generation. Depletion of ECSIT or TRAF6 in macrophages results in reduced mROS production and impaired ability to kill intracellular bacteria. Additionally, reducing mROS by expressing catalase in mitochondria also impairs bacterial killing. These findings reveal a novel pathway linking innate immune signaling to mitochondria and highlight the importance of mROS in antibacterial responses.