Mycotoxin affects the world__ampersandsignrsquo;s food crops and creates a large economical loss in the developed and developing countries. Aflatoxins are a group of mycotoxins that mainly produced by Aspergillus species viz., A. flavus, and A. parasiticus. An aflatoxins contamination of maize grains has exhibiting a serious threat to human and animal health over the past two decades. Toprotect the safety of food commodities, regular monitoring and diagnosis of the presence and amount of non-permissible levels of aflatoxins in food is necessary to take appropriate management measures. Maize grain samples were collected from Ilu Galan and Bako districts of West Shoa and Gobu Sayo district of East Wollega zones of Oromiya; from different grain storage types. About 500 gr of maize grains were sampled from each sampling spot. PDA media was used for isolation of associated maize grains sample associated fungi. Sun-culturing and purification of the associated fungi were done and preserved using agar slant technique. The associated fungal mycoflora were characterized based on morphological and growth sporulation properties. Enzyme Linked Immuno Sorbent Assay (ELISA) diagnostic kit were used for identification and quantification of aflatoxins. Aspergillus, Fusarium Penicillium and Trichoderma species were identified and characterized. Aflatoxin B1 was identified and quantified from zero to 381.6__ampersandsignmu;g/kg. About 34.4% of the samples were positive to aflatoxin B1 compared to Food and Drug Administration (20__ampersandsignmu;g/kg) and European Union (4__ampersandsignmu;g/kg), respectively. The management of mycotoxigenic fungi, improvement of storage methods, development of resistant maize varieties and awareness creations could be possible solutions