Peroxynitrite anion (ONOO−), a potent oxidant formed by the fast reaction of nitric oxide (•NO) with superoxide radical (O2•−), is increasingly recognized as a key downstream effector in NADPH oxidase (Nox)-dependent pathways. In physiological and pathological contexts where Nox—such as Nox1, Nox4 and Nox2—are activated, formation becomes highly favored, particularly when nitric oxide synthase activity co-occurs. The production of peroxynitrite in these settings contributes to both host defense and tissue injury. For example, in innate immune responses, Nox2-derived O2•− combines with •NO in phagosomes to generate peroxynitrite, supporting microbial killing. However, sustained Nox activation, as observed in chronic inflammation, cardiovascular disease, or neurodegeneration, can lead to excessive peroxynitrite formation and oxidative damage. This is largely due to the reactivity of peroxynitrite and its derived species: nitrogen dioxide (•NO2) and carbonate radicals (CO3•−); which target proteins, lipids, and nucleic acids, affecting cellular integrity and signaling. Importantly, peroxynitrite can mediate selective modifications such as protein tyrosine nitration, which often serves as a biomarker of nitro-oxidative stress. Understanding the interplay between Nox activity, peroxynitrite generation, and antioxidant defenses is crucial to defining its dual role as a cytotoxic and regulatory molecule in Nox-driven pathophysiological processes.