Reactive oxygen species (ROS) are essential components of multiple cellular pathways, but excessive or improperly localized ROS can damage cells. ROS function as antimicrobial effector molecules and signaling molecules that regulate processes such as NF-κB transcriptional activity and the production of DNA-based neutrophil extracellular traps (NETs). The main sources of cellular ROS are mitochondria and NADPH oxidases (NOXs). While initially considered by-products of oxidative metabolism, mitochondrial ROS (mtROS) are now known to be involved in signaling pathways, including those regulating immune responses and autophagy. Mitochondria act as metabolic hubs, integrating these pathways with the cell's metabolic state. ROS and mitochondria are thus a nexus of multiple pathways that determine the response of cells to disruptions in cellular homeostasis, such as infection, sterile damage, and metabolic imbalance. This review discusses the roles of mitochondria in generating ROS-derived antimicrobial effectors, the interplay of mitochondria and ROS with autophagy and NET formation, and the activation of the NLRP3 inflammasome by ROS and mitochondria. While the contributions of NOX-generated ROS to these processes are described, the focus is on the emerging roles of mtROS. ROS play a critical role in antimicrobial defense, including the oxidative burst in phagocytes, leading to the production of hypochlorous acid and increased intraphagosomal pH. ROS are also involved in autophagy, with mtROS contributing to the degradation of damaged mitochondria. The NLRP3 inflammasome is a sensor of homeostatic disruptors, activated by ROS and mitochondrial signals. ROS and mtROS are involved in the activation of the NLRP3 inflammasome through various mechanisms, including the regulation of NLRP3 by thioredoxin-interacting protein (TXNIP) and Nrf2. Mitochondria also contribute to NLRP3 activation through the release of mitochondrial DAMPs and the production of mtROS. The interplay between ROS, mitochondria, and autophagy is complex and involves multiple pathways, including the regulation of NLRP3 by mitochondrial metabolites and DAMPs. Understanding these interactions is crucial for elucidating the mechanisms of immune responses and inflammatory diseases.Reactive oxygen species (ROS) are essential components of multiple cellular pathways, but excessive or improperly localized ROS can damage cells. ROS function as antimicrobial effector molecules and signaling molecules that regulate processes such as NF-κB transcriptional activity and the production of DNA-based neutrophil extracellular traps (NETs). The main sources of cellular ROS are mitochondria and NADPH oxidases (NOXs). While initially considered by-products of oxidative metabolism, mitochondrial ROS (mtROS) are now known to be involved in signaling pathways, including those regulating immune responses and autophagy. Mitochondria act as metabolic hubs, integrating these pathways with the cell's metabolic state. ROS and mitochondria are thus a nexus of multiple pathways that determine the response of cells to disruptions in cellular homeostasis, such as infection, sterile damage, and metabolic imbalance. This review discusses the roles of mitochondria in generating ROS-derived antimicrobial effectors, the interplay of mitochondria and ROS with autophagy and NET formation, and the activation of the NLRP3 inflammasome by ROS and mitochondria. While the contributions of NOX-generated ROS to these processes are described, the focus is on the emerging roles of mtROS. ROS play a critical role in antimicrobial defense, including the oxidative burst in phagocytes, leading to the production of hypochlorous acid and increased intraphagosomal pH. ROS are also involved in autophagy, with mtROS contributing to the degradation of damaged mitochondria. The NLRP3 inflammasome is a sensor of homeostatic disruptors, activated by ROS and mitochondrial signals. ROS and mtROS are involved in the activation of the NLRP3 inflammasome through various mechanisms, including the regulation of NLRP3 by thioredoxin-interacting protein (TXNIP) and Nrf2. Mitochondria also contribute to NLRP3 activation through the release of mitochondrial DAMPs and the production of mtROS. The interplay between ROS, mitochondria, and autophagy is complex and involves multiple pathways, including the regulation of NLRP3 by mitochondrial metabolites and DAMPs. Understanding these interactions is crucial for elucidating the mechanisms of immune responses and inflammatory diseases.