Fungi and mycotoxin contamination of feed poses a significant risk to broiler chickens (BC) and humans, as well as lowering feed quality. This study assessed fungal contamination of BC rations at Sohag Governorate, Egypt, throughout the year and evaluated aflatoxigenicity (AFTG) of Aspergillus flavus (A. flavus) isolates as well as antifungal activity (AA) of curcumin nanoparticles (Cur-NPs) and Saccharomyces cerevisiae (S. cerevisiae) against aflatoxigenic (AFT) A. flavus isolates. Mycological examination revealed that 92.9% of the investigated rations were contaminated by fungi, and total mould count ranged from 1 × 10 to 1.91 × 107 CFU/g of ration. Isolated fungi belonged to 27 fungal genera, with predominance of genus Aspergillus followed by Penicillium, Mucor, Cladosporium, Eurotium, and Scopulariopsis. Furthermore, Aspergillus isolates were morphologically characterized into 20 different species, with predominance of A. flavus followed by Aspergillus niger, Aspergillus terreus, and Aspergillus ustus, respectively. Also, mycological examination revealed that fungal contamination of the rations was the highest in autumn, followed by summer, winter, and spring, respectively. Statistically, there were significant differences in the incidences of different fungal genera and Aspergillus spp. between the different year seasons (P < 0.001). Regarding AFTG of A. flavus isolates, 27.3% of them were AFT with variable degrees, and 90% of the strong AFT isolates were AFB1 producers, with high amounts ranging from less than 25 to 71 µg/L. Statistical analysis revealed the presence of a significant difference in the distribution of AFT A. flavus isolates through the different seasons (P < 0.001). PCR revealed that 100% and 30% of the investigated strong AFT A. flavus isolates (n = 10) harboured nor-1 and ver-1 genes, respectively. On the other hand, Cur-NPs exhibited strong AA against AFT A. flavus isolates, which increased with increasing Cur-NPs concentration. Also, the growth of all AFT A. flavus isolates was inhibited with S. cerevisiae, with an inhibition zone of 24.6 ± 0.64 mm2. Therefore, immediate essential control and preventive measures are needed to ensure food safety. Cur-NPs and S. cerevisiae are recommended for evaluation and use as effective fungicides to control AFT A. flavus and its potential for aflatoxin production in BC feeds.