Fungal contamination in nuts is unavoidable and is a major challenge to nuts safety, quality and then for human health after consumption particularly immunocompromised individuals. This study aimed to early detection of potential pathogenic Aspergillus in nuts to avoid the harmful effects of allergens and mycotoxins. Three different geographic locations in Basra, Iraq were screened, and a total of 28 fungal isolates were recovered from tested nuts including cashews, almonds, walnuts, pistachio and sunflower seeds. ELISA was used to identify isolated fungi based on antigenic structure via using a highly specific MAb for Aspergillus, JF5. The accuracy of ELISA was validated by ITS-rDNA sequence in comparison with NCBI database. The highest incidence of isolated fungi belongs to main four genera Aspergillus (78.57%), Alternaria (10.71%), Cladosporium (7.14%), followed by Penicillium (3.75%). The most dominant species was A. flavus, a potential aflatoxins producer, (28.57%). Abu was highly contaminated geographic site comparing with Zubayr and Qarmat. These findings raise awareness about contaminated nuts particularly there is no database available and highlighted the accuracy of using specific MAb for fungal identification and diagnosis. The occurrence of well-known toxigenic species such as A. niger, A. flavus and A. terreus suggested the possible risk of mycotoxin contamination of the nuts for human consumption.