The modern utilization of essential oils such as ginger oil (GO) as an anti-aflatoxin represents a potential target for food preservation and safety; however, the mechanism of action is still unclear. Nanoemulsions, through an edible coating, can enhance the oil’s bioactivity, increase its hydrophilicity, and extend the final product’s shelf-life. In the present study, two edible films for the GO nanoemulsion were prepared by ultrasonication using carboxymethyl cellulose (FB1-GO) and sodium alginate (FB2-GO). The droplet size of FB2-GO was finer (126.54 nm) compared to FB1-GO (289.77 nm). Meanwhile, both had high stability proved by z-potential; +31.54 mV (FB1-GO) and +46.25 mV (FB2-GO) with low PDI values (<0.4). Using gas chromatography-mass spectrometry, the hydrodistilled GO showed 25 compounds, representing 99.17% of the total oil, with α-zingiberene (29.8%), geranial (10.87%), β-bisabolene (8.19%), and ar-curcumene (5.96%) as the predominant. A dramatic increase in α-zingiberene, α-bisabolene and ar-curcumene was due to the homogenization conditions in both FB1-GO and FB2-GO compared to the GO. The FB1-GO exhibited superior antibacterial activity against the examined strains of bacterial pathogens, while FB2-GO was more effective as an antifungal agent on the tested Aspergillus fungi strains. In a simulated liquid media, FB2-GO inhibited the total growth of fungi by 84.87–92.51% and showed the highest reduction in the aflatoxin amount produced. The in silico study presented that, among the GO volatile constituents, sesquiterpenes had the highest binding free energies against the enzymes responsible for aflatoxin production compared to monoterpenes. α-Bisabolene showed the highest affinity toward polyketide synthase (−7.5 Kcal/mol), while ar-curcumene was the most potent against cytochrome P450 monooxygenase (−8.3 Kcal/mol). The above findings clarify the reasons for aflatoxin reduction in simulated media during incubation with FB1-GO and FB2-GO.