Beta-lapachone (1), a natural ortho-naphthoquinone, has been widely used for its large pharmacological activities, particularly against cancers. In the present study, amperometry at platinized carbon microelectrode was used to investigate the activity of (1), at different concentrations after various incubation times, on the oxidative bursts release of single macrophages. The results show that the presence of 0.1 to 100 µM of beta-lapachone, within one hour of incubation, leads to a decrease of reactive oxygen and nitrogen species release, comparatively to control. Conversely, when the incubation time increases (4 hours and more), the quantity of the species released in presence of 1 µM of (1) increases. Moreover, macrophage membranes became withered after 4 hours of incubation in presence of 10 µM of (1) and leading to cell death. These studies demonstrated the advantage of electrochemical methods for analyzing in real-time and quantitatively the effect of beta-lapachone on oxidative burst.