This study reveals a novel pathway linking innate immune signaling to mitochondria, demonstrating that Toll-like receptor (TLR) signaling enhances macrophage bactericidal activity through mitochondrial reactive oxygen species (mROS). TLR engagement, particularly by TLR1, TLR2, and TLR4, recruits mitochondria to phagosomes and increases 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 (ESIT), a protein involved in mitochondrial respiratory chain assembly. TRAF6 promotes the ubiquitination and enrichment of ESIT at the mitochondrial periphery, leading to increased mitochondrial and cellular ROS generation. Macrophages deficient in TRAF6 or ESIT exhibit reduced TLR-induced ROS and impaired bacterial killing. Additionally, reducing mROS by expressing catalase in mitochondria results in defective bacterial killing, confirming the role of mROS in bactericidal activity. These findings highlight mitochondria as hubs for innate immune signaling and underscore the importance of mROS in antibacterial responses. The study also reveals a remarkable symmetry between mitochondrial antiviral signaling protein (MAVS) and ESIT in innate immune responses. The results demonstrate that TRAF6-ECSIT signaling is engaged downstream of bacterial PAMP-sensing TLRs for robust ROS production, similar to MAVS signaling in virus-sensing RIG-I-like receptors for type I interferon production. The study further supports the emerging idea that mitochondria serve as hubs for innate immune signaling and the generation of effector responses.This study reveals a novel pathway linking innate immune signaling to mitochondria, demonstrating that Toll-like receptor (TLR) signaling enhances macrophage bactericidal activity through mitochondrial reactive oxygen species (mROS). TLR engagement, particularly by TLR1, TLR2, and TLR4, recruits mitochondria to phagosomes and increases 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 (ESIT), a protein involved in mitochondrial respiratory chain assembly. TRAF6 promotes the ubiquitination and enrichment of ESIT at the mitochondrial periphery, leading to increased mitochondrial and cellular ROS generation. Macrophages deficient in TRAF6 or ESIT exhibit reduced TLR-induced ROS and impaired bacterial killing. Additionally, reducing mROS by expressing catalase in mitochondria results in defective bacterial killing, confirming the role of mROS in bactericidal activity. These findings highlight mitochondria as hubs for innate immune signaling and underscore the importance of mROS in antibacterial responses. The study also reveals a remarkable symmetry between mitochondrial antiviral signaling protein (MAVS) and ESIT in innate immune responses. The results demonstrate that TRAF6-ECSIT signaling is engaged downstream of bacterial PAMP-sensing TLRs for robust ROS production, similar to MAVS signaling in virus-sensing RIG-I-like receptors for type I interferon production. The study further supports the emerging idea that mitochondria serve as hubs for innate immune signaling and the generation of effector responses.