Aspergillus flavus is an important pathogenic fungus affecting many crops and is one of the main sources of their aflatoxin contamination. The primary method of limiting this pathogen is using chemical fungicides, but researchers focus on searching for other effective agents for its control due to many disadvantages and limitations of these agrochemicals. The results obtained in the present study indicate the high potential of two yeast strains, Aureobasidium pullulans PP4 and A. pullulans ZD1, in the biological control of A. flavus. Under in vitro conditions, mycelial growth was reduced by 53.61% and 63.05%, and spore germination was inhibited by 68.97% and 79.66% by ZD1 and PP4 strains, respectively. Both strains produced the lytic enzymes chitinase and β-1,3-glucanase after 5 days of cultivation with cell wall preparations (CWP) of A. flavus in the medium as a carbon source. In addition, the tested yeasts showed the ability to grow over a wide range of temperatures (4–30 °C), pH (4–11), and salinity (0–12%) and showed tolerance to fungicides at concentrations corresponding to field conditions. Both isolates tested were highly tolerant to cupric oxychloride, showing biomass gains of 85.84% (ZD1) and 87.25% (PP4). Biomass growth in the presence of fungicides azoxystrobin was 78.71% (ZD1) and 82.65% (PP4), while in the presence of difenoconazole, it was 70.09% (ZD1) and 75.25% (PP4). The yeast strains were also tested for antagonistic effects against A. flavus directly on tomato fruit. Both isolates acted effectively by reducing lesion diameter from 29.13 mm (control) to 8.04 mm (PP4) and 8.83 mm (ZD1)