Chillies are a very important cash crop of Pakistan. The effects of gamma irradiation on microbial load, aflatoxin B 1 (AFB 1 ) and total aflatoxins have been studied in chillies samples, collected from different districts of Punjab, Pakistan. Aflatoxins were analyzed using HPLC equipped with a fluorescence detector. The results revealed that among the Aspergillus species isolated, those belonging to section parasiticus were predominant. Gamma radiations of doses 2, 4 and 6kGy were employed on fungi and chilli samples. The results have demonstrated that the dose of 6kGy reduced the fungal load by 5logs. Furthermore, 6kGy reduced the level of AFB 1 and total AFs in ground and whole chillies by 1–2logs (α < 0.05).