The uncertainty about the possible involvement of a luciferase in fungal bioluminescence has not only hindered the understanding of its biochemistry but also delayed the characterization of its constituents. The present report describes how in vitro light emission can be obtained enzymatically from the cold and hot extracts assay using different species of fungi, which also indicates a common mechanism for all these organisms. Kinetic data suggest a consecutive two-step enzymatic mechanism and corroborate the enzymatic proposal of Airth and Foerster. Finally, overlapping of light emission spectra from the fungal bioluminescence and the in vitro assay confirm that this reaction is the same one that occurs in live fungi.