Abstract Fungal infection and mycotoxin contamination in fresh and stored kernels of walnut ( Juglans regia) collected from different localities of Uttaranchal (India) were investigated. Fresh samples carry a combination of field as well as storage fungi. Species of Alternaria, Aspergillus and Penicillium were predominant. Thirty-nine percent of Aspergillus flavus isolates were toxigenic and produced up to 2170 μg/l of aflatoxin B 1 in the liquid media. Aflatoxin B 1 was the most common mycotoxin encountered as a natural contaminant in the stored samples. Twenty-one percent of fresh samples contained aflatoxin B 1 in low concentrations. The concentration of aflatoxin B 1 in fresh as well as stored samples was in the range of 140–1220 μg/kg. Characteristic rotting was observed in fresh as well as stored samples. The walnut kernels exhibited significant reductions in the levels of oil, starch and protein content during fungal infection.