Reactive oxygen species (ROS) play crucial roles in multiple cellular pathways, including immune responses and autophagy. Mitochondria and NADPH oxidases (NOXs) are the primary sources of cellular ROS. While NOX-generated ROS have been extensively studied, recent evidence suggests that mitochondrial ROS (mtROS) are also important in immune responses and signaling pathways. MtROS are produced in the mitochondria and can be regulated by immune signaling pathways, contributing to immune responses such as bactericidal activity, autophagy, and inflammation. The review discusses the roles of mitochondria in generating ROS-derived antimicrobial effectors, the interplay between mitochondria, ROS, and autophagy, and the activation of the NLRP3 inflammasome by ROS and mitochondria. It highlights the complex mechanisms involving ROS and mitochondria in these processes, including the involvement of mtROS in priming and activation steps of the NLRP3 inflammasome. The review also explores the links between ROS, mtROS, and NLRP3 activation, including the role of thioredoxin-interacting protein (TXNIP) and the transcription factor Nrf2. Additionally, it discusses the additional roles of mitochondria and mtROS in activating the NLRP3 inflammasome through mitochondrial DAMPs and metabolites. The unified model of NLRP3 inflammasome activation suggests that mitochondria serve as a signaling platform and source of DAMPs, contributing to the diverse stimuli that activate the inflammasome.Reactive oxygen species (ROS) play crucial roles in multiple cellular pathways, including immune responses and autophagy. Mitochondria and NADPH oxidases (NOXs) are the primary sources of cellular ROS. While NOX-generated ROS have been extensively studied, recent evidence suggests that mitochondrial ROS (mtROS) are also important in immune responses and signaling pathways. MtROS are produced in the mitochondria and can be regulated by immune signaling pathways, contributing to immune responses such as bactericidal activity, autophagy, and inflammation. The review discusses the roles of mitochondria in generating ROS-derived antimicrobial effectors, the interplay between mitochondria, ROS, and autophagy, and the activation of the NLRP3 inflammasome by ROS and mitochondria. It highlights the complex mechanisms involving ROS and mitochondria in these processes, including the involvement of mtROS in priming and activation steps of the NLRP3 inflammasome. The review also explores the links between ROS, mtROS, and NLRP3 activation, including the role of thioredoxin-interacting protein (TXNIP) and the transcription factor Nrf2. Additionally, it discusses the additional roles of mitochondria and mtROS in activating the NLRP3 inflammasome through mitochondrial DAMPs and metabolites. The unified model of NLRP3 inflammasome activation suggests that mitochondria serve as a signaling platform and source of DAMPs, contributing to the diverse stimuli that activate the inflammasome.