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The inhibitory action of 20 antagonistic <i>Trichoderma</i> isolates against the aflatoxigenic isolate <i>A. flavus</i> ITEM 9 (Af-9) and their efficacy in reducing aflatoxin formation in vitro were examined. Production of metabolites with inhibitory effect by the <i>Trichoderma</i> isolates was also investigated. Antagonistic effect against Af-9 was assessed by inhibition of radial growth of the colonies and by fungal interactions in dual confrontation tests. A total of 8 out of 20 isolates resulted in a significant growth inhibition of 3-day-old cultures of Af-9, ranging from 13% to 65%. A total of 14 isolates reduced significantly the aflatoxin B₁ (AfB₁) content of 15-day-old Af-9 cultures; 4 were ineffective, and 2 increased AfB₁. Reduction of AfB₁ content was up to 84.9% and 71.1% in 7- and 15-day-old cultures, respectively. Since the inhibition of Af-9 growth by metabolites of <i>Trichoderma</i> was not necessarily associated with inhibition of AfB₁ production and vice versa, we investigated the mechanism of reduction of AfB₁ content at the molecular level by examining two strains: one (T60) that reduced both growth and mycotoxin content; and the other (T44) that reduced mycotoxin content but not Af-9 growth. The expression analyses for the two regulatory genes <i>aflR</i> and <i>aflS</i>, and the structural genes <i>aflA</i>, <i>aflD</i>, <i>aflO</i> and <i>aflQ</i> of the aflatoxin biosynthesis cluster indicated that neither strain was able to downregulate the aflatoxin synthesis, leading to the conclusion that the AfB₁ content reduction by these <i>Trichoderma</i> strains was based on other mechanisms, such as enzyme degradation or complexation. Although further studies are envisaged to identify the metabolites involved in the biocontrol of <i>A. flavus</i> and prevention of aflatoxin accumulation, as well as for assessment of the efficacy under controlled and field conditions, <i>Trichoderma</i> spp. qualify as promising agents and possible alternative options to other biocontrol agents already in use.