Aspergillus flavus is a fungal pathogen of crops including maize and is frequently associated with the accumulation of aflatoxin, which presents a concern for human and animal health. In maize, good agricultural practices can only partially reduce Aspergillus occurrence, and biological control through atoxigenic strains decreases aflatoxin content, but increasing mold decreases kernel quality. The control of Aspergillus infections by chemicals is not authorized in maize, but recently in Italy, some sterol-biosynthesis inhibitors obtained an emergency authorization for use against Fusarium infections. Therefore, it could be interesting to determine if these inhibitors could also be useful against A. flavus. A mixture of prothioconazole and tebuconazole was tested in vitro on toxigenic and atoxigenic strains and on populations with respect to conidial germination, germinal potential and fungal growth. The fungicide mixture inhibited fungal growth, with a higher effect on toxigenic strains than on atoxigenic strains. All considered parameters were significantly reduced for monoconidial strains at doses lower than those recommended for field application for straw cereals; however, under-dosing should be avoided since it increases aflatoxin production. Field trials under natural infection conditions revealed the efficacy of the fungicide treatment on the reduction of both Aspergillus occurrence and aflatoxin accumulation in maize. In a context in which the sustainable use of pesticides can be guaranteed, our findings support the inclusion of azole-based fungicides in a multiple-strategy approach to control Aspergillus infections in maize.