THIRTY wheat flour samples were collected from different bakeries and markets in Qena City, Egypt over five months were used to measure moisture content, fungi and aflatoxins. Moisture contents ranged from 5.92% to 14.43%. Twenty-one fungal species belonging to 13 genera were isolated from the wheat flour samples on Czapeks agar media at 28ᵒC. The most common fungal genera were Aspergillus, Mucor and Penicillium. The correlation coefficient analysis revealed to strong positive correlation between moisture content and average total count of fungi in wheat flour samples (0.92). Qualitative and quantitative determination of aflatoxins in 29 isolates of the A. flavi group (A. flavus, A. flavus var. columnaris and A. parasiticus) were made using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), respectively. The results for (TLC) were as follow: Fourteen isolates (48.2%) had the ability to produce aflatoxin B1, five isolates (17.2%) could produce both aflatoxin B1 and aflatoxin B2 and ten (34.4%) isolates were negative. By using HPLC, the concentrations of aflatoxin were 13.416–9229.343, 2.639–152.668 and 6.391–79.507μg/L for AFB1, AFB2, and AFG2, respectively.