A novel oxygen-based microplate assay for studying fungal activity is described. Fungal activity results in a change of oxygen concentration in CVC-96 plates and thus produces a signal that enables continuous monitoring of fungal activity. In this study the oxygen consumption was different for three tested fungi, Fusarium oxysporum f. sp. lycopersici, Verticillium dahliae and Trichoderma longibrachiatum. The assay described is a highly sensitive and reliable method for monitoring fungal activity. This assay does not interfere with fungal development and there is no need to kill the cells to take a measurement. This test would be useful for studying reactions that consume oxygen so possible applications of this test could be physiological studies, testing of fungicidal or fungistatic compounds, and studying enzyme reactions. The system provides a valuable insight into the kinetics of fungal activity and is suitable to test other systems.