Aflatoxins (AFs) are chemical compounds with high toxic impacts produced by Aspergillus section Flavi. Toxigenic fungi contaminate foods and feed, thus presenting biological and chemical contamination resulting in health issues. Therefore, this study aims to evaluate the prevalence of toxigenic fungi in several cereal grains collected from different Egyptian governorates, as well as to evaluate the ability of toxigenic fungi to produce AFs using coconut agar medium (CAM), thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC). Results revealed that Aspergillus spp. were the predominant fungi in barley, white corn, yellow corn, and rice samples, followed by Penicillium spp. Data also showed that barley, white corn, yellow corn, and rice obtained from Alexandria governorate were highly contaminated, whereas wheat obtained from Beheira governorate was highly contaminated. On studying the ability of A. parasiticus, and A. flavus to produce AFs on CAM, 63.15% of the fungal isolates gave different blue and green fluorescence densities under a UV lamp at the reverse sides of the CAM plates. The fungal isolates showing fluorescence were further determined using TLC, whereas twenty-two fungal isolates were tested positive for the AFs production. The HPLC analyses revealed that twenty-one out of twenty-two fungal isolates produced four AF types of AFs. Detectable aflatoxin B1 (AFB1) concentrations ranged between 0.02 and 875.03 ng/g; 0.07 and 13.55 ng/g for aflatoxin B2 (AFB2); 0.06 and 2.85 ng/g for aflatoxin G1 (AFG1); and 0.71 and 89.29 ng/g for aflatoxin G2 (AFG2). These results indicated the importance of a control system to regulate fungal and mycotoxin production in grains before their consumption or manufacturing to limit AFs contamination.