A great diversity of agricultural products is susceptible to contamination caused by Aspergillus flavus . To reduce fungal contamination, the application of natural products has been proposed, including chitosan and propolis, due to its broad and recognized antimicrobial activity on several microorganisms. Currently, the application of nanotechnology allows for a greater activity to be more reactive and efficient. The objectives of this research were to characterize by TEM and Z potential some of the studied nanoparticles and to determine the in vitro antifungal activity of the formulations and the production of aflatoxins of the treated fungus. For this, individual treatments and different nanoformulations were elaborated by varying the percentage of the components such as chitosan solution, chitosan nanoparticles, an extract of propolis, nanoparticles of propolis, glycerol and canola oil. The final concentrations of the formulations were of 20%, 30% and 40% and the control consisted of Czapeck-dox agar medium. TEM micrographies showed a spherical morphology in a range of 2.3–3.0 nm with values of Z potential from 18.5 to 116.2 nm. Compared to the untreated fungus, the highest effect was seen in the parameter of spore germination, since inhibition was of c. a. 97% corresponding to the formulation containing chitosan + propolis nanoparticles + chitosan nanoparticles + propolis extract at the highest concentration of 40%. At this same concentration, the production of aflatoxins was 100% inhibited with the treatment with chitosan at 1%. Since these results are under carefully controlled conditions, further research should be extended to different fruit and vegetables affected by this fungus.