Oxidative stress plays an important role in chronic respiratory diseases where the use of non-invasive ventilation seems to reduce the oxidative damage. Data on acute respiratory failure are still lacking. The aim of the study is to investigate the interplay between oxidative stress and acute respiratory failure, and the role of non-invasive ventilation in this setting. We enrolled 60 patients suffering from acute respiratory failure (PaO/FiO ratio <300): 30 consecutive patients treated with non-invasive ventilation and 30 consecutive patients treated with conventional oxygen therapy. Serum levels of soluble Nox2-derived peptide (sNOX2-dp), a marker of NADPH-oxidase activation, and 8-iso-PGF2α and HO, markers of oxidative stress, were evaluated at baseline and after 3 h of treatment. At baseline, higher values of sNOX2-dp, 8-iso-PGF2α and HO are associated with lower values of PaO/FiO ratio (p < 0.001). After 3 h, serum levels of sNOX2-dp, HO, and 8-iso-PGF2α significantly decrease in patients treated with non-invasive ventilation, but not in patients treated with conventional oxygen therapy. Delta changes of oxidative stress parameters correlate inversely with the delta changes of PaO/FiO (R = -0.623, p < 0.001 for sNOX2-dp; R = -0.428, p < 0.001 for HO; R = -0.548, p < 0.001 for 8-iso-PGF2α). In the acute respiratory failure setting, treatment with non-invasive ventilation reduces the levels of oxidative stress in the first hours. This reduction is associated with an improvement of PaO/FiO ratio as well as in a reduction of NADPH-oxidase activity.