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Aflatoxin contamination in food commodities is a major food safety concern all over the world. These aflatoxins are a group of carcinogenic secondary metabolites produced by some fungi belonging to Aspergillus section Flavi. To minimize the level of aflatoxins in food commodities, a wide range of pre- and post-harvest procedures have been employed. In this study, aqueous extracts of 13 medicinal herbs were assessed for their ability to detoxify aflatoxin B1 (AFB1), the most potent and commonly occurring carcinogen in foods. Among them, herbal extracts of Hybanthus enneaspermus, Eclipta prostrata and Centella asiatica exhibited over 70% detoxification of AFB1. The degradation of AFB1 upon treatment with these herbal extracts was confirmed by liquid chromatography–mass spectrometry analysis. Two fractions (Rf 0.75 and 0.87) purified from H. enneaspermus by using thin-layer chromatography displayed in vitro AFB1-detoxifying properties. Gas chromatography–mass spectrometry analysis of the active fractions revealed the presence of linalool and bornyl acetate as the major components suggesting possible involvement of volatile compounds of H. enneaspermus in the detoxification of AFB1. To our knowledge, this is the first report on the detoxification of AFB1 by H. enneaspermus, E. prostrata and C. asiatica aqueous extracts.