The present study reports the antifungal, aflatoxin B inhibitory, and free radical scavenging activity of chitosan-based nanoencapsulated Bunium persicum Boiss. essential oil (Ne-BPEO). The chemical profile of BPEO was identified through Gas chromatography mass spectrometry analysis where cuminaldehyde (21.23%), sabinene (14.66%), and γ-terpinen (12.49%) were identified as the major compounds. Ne-BPEO was prepared using chitosan and characterized by Scanning electron microscope (SEM), Atomic force microscope (AFM), Fourier-transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) assay. Ne-BPEO completely inhibited the growth and aflatoxin B production at a concentration of 0.3 μL/mL. The antifungal and aflatoxin B inhibitory effects were related to decreasing in ergosterol content, leakage of membrane ions (Ca, K, and Mg), impairment in carbohydrate catabolism, and functioning of ver-1 gene of A. flavus exposed to Ne-BPEO over the control. In addition, Ne-BPEO exhibited promising free radical scavenging activity through DPPH assay (IC 12.64 μL/mL) with high thermo-stability. Therefore, chitosan could be used as a carrier agent of plant-based preservative to enhance the shelf-life of food products against A. flavus and aflatoxin B contamination.