Hygienic quality of Tunisian pearl millet flour is always of major concern to consumers as well as all involved in the production, processing and distribution sectors. In the present study, the microbiological and biochemical properties of this food were examined following gamma-radiation. The D10-values for the Total Aerobic Plate Count, yeasts and moulds were respectively 1.5 and 3.7 kGy. Furthermore, millet flour is commonly susceptible to mycotoxin contaminations, so the Ochratoxin A residues were also investigated; a reduction of 74% was observed with 10 kGy. Moreover, the radiation process did not significantly alter fatty acids composition of the millet flour as obtained with Gas chromatography-flame ionisation detector technic. The peroxide value had increased from 26.16 to 34.43 meq O2/kg with 3 kGy. At 1 kGy, we noticed an important loss of vitamin A of about 88.6%. In contrast, the total phenolic content, the ABTS-RSA and the DPPH-RSA of the radiated millet flour exhibited non-significant changes (p < 0.05).