One strain of Aspergillus flavus ATCC 26949 and one strain of Fusarium graminearum ZMPBF 1244 were used to investigate the effect of growth in mixed culture on aflatoxin B1 (AFB1) accumulation in respect to time and temperature of cultivation in vitro. The experiments were carried out with aflatoxigenous mould A. flavus ATCC 26949 in pure culture and in mixed culture, respectively, the latter with the common mould F. graminearum ZMPBF 1244. F. graminearum does not produce aflatoxins and chromatographycally similar compounds. The amount of biomass was estimated by measuring the chitin content; the aflatoxin accumulation by means of UV light and ELISA method. It was established that the accumulation of AFB1 primarily depend on the temperature of cultivation, rather than on growth of the mycelium. The biomass of the mixed culture of A. flavus and F. graminearum after 35 days of cultivation reduces the amount of AFB1 by 55 to 76% in respect to highest values obtained in experiments with the pure culture of A. flavus. Decrease of this toxin amount is more pronounced in the substrate at the higher water content and at the higher temperature of cultivation. As a general rule, cultures grown in mixed culture under investigate temperature conditions produced less amount of AFB1 than A. flavus grown in pure culture.