Fungal contamination in poultry farms poses significant hazards to broiler health, production and public health, considering the fact that the poultry industry represents a significant role in Egypt's economy and food security. The total fungal count (TFC), mold species prevalence, antifungal susceptibility patterns, and virulence genes of Aspergillus flavus isolated from broiler farms in Egypt's Sharkia Governorate were the main objectives of this study. In the summer and winter of 2025, 324 samples were taken from three commercial broiler farms. Cloacal swabs, air, water, litter, feed, workers' hands, equipment and wall swabs were among the investigated samples. Sabouraud Dextrose Agar was used in order to determine total fungal count (TFC) and microscopic and standardized culture methods were used to identify mold isolates. Two A. flavus isolates were assessed for their antifungal susceptibility using broth microdilution, and multiplex PCR was used to identify the virulence genes nor-1, ver-1 and omtA. The findings showed that the mean fungal load was highest in litter samples (6.24±0.35 log CFU/g) and lowest in air samples (3.68±0.32 log CFU/m³). Seasonal variation showed slightly higher contamination in summer. The most prevalent isolates were Aspergillus species, particularly A. flavus and A. ochraceus (12.5% each). Amphotericin-B had the most significant MIC values, while Luliconazole and lanoconazole demonstrated the strongest antifungal activity with the lowest MIC values. PCR verified that the isolates under investigation included aflatoxigenic genes. For the purpose to lower fungal contamination and related health hazards, these findings highlight poultry litter as the main fungal reservoir and emphasize the need for strict hygiene, biosecurity and ongoing monitoring.