Mitochondria are key regulators of immune cell function, going beyond their traditional role in ATP and metabolite production to support anabolic processes and act as hubs for intracellular signaling. A key aspect of this signaling function is the production of mitochondrial reactive oxygen species (mtROS), which act as critical second messengers in both adaptive and innate immune regulation. Immune cells maintain an optimal concentration of mtROS to maintain physiological responses, and excessive or lack of mtROS production contributes to chronic inflammation, autoimmunity, and cancer. Here, we review the molecular mechanisms controlling mtROS production and detoxification, their role in shaping macrophage and T cell fate and function, and their implications for disease pathogenesis.