Mycotoxins are toxic metabolites mainly produced by Aspergillus spp. fungi. Further fungal characterization and mycotoxin estimation in feed are crucial measures for animal health and safety. The aim of this study was to identify and characterize mycotoxin-producing fungi in poultry feed. Forty poultry ration samples were collected and were subjected to morphological and molecular as well as to phylogenetic analyses of Aspergillus section Nigri. On average, 40% (16 of 40 samples) were contaminated with Aspergillus section Nigri fungi. Moreover, A. niger and A. tubingensis were identified and characterized in the contaminated poultry feed using molecular and phylogenetic analyses. Furthermore, our results showed that ochratoxin A was produced by some fungal isolates indicating their mycotoxigenicity. In conclusion, A. niger and A. tubingensis contaminate poultry feed and can produce mycotoxins under optimal conditions. Furthermore, several preventive and control measures should be undertaken to reduce the occurrence of fungal contamination and toxin production in poultry rations.