The metabolic activity of the aflatoxigenic fungus, Aspergillus flavus co-cultured with the biocontrol yeast, Pichia anomala was examined using several viability stains. Both the FUN-1 stain and the combined use of DiBAC(4)(5) with CDFA-AM stains were applied in this study. The results suggest that the ATP-generating system in A. flavus was inactivated as the ratio of yeasts to fungi increased in the dual culture. A decrease in hyphal membrane potential and esterase activity was substantiated by the combined stains of DiBAC(4)(5) and CDFA-AM. Reduced metabolic function in conjunction with cell wall damage of A. flavus hindered the growth and biomass production of this fungus. Viability stains such as FUN-1 and DiBAC(4)(5) with CDFA-AM may assist in elucidating the biocontrol mechanism by allowing for the visualization of the antagonistic effect of yeast species on target fungi in situ, as well as for screening potent biocontrol yeast agents against fungal pathogens.