| Summary: | The present study deals with the encapsulation of <i>Cymbopogon khasiana</i> × <i>Cymbopogon pendulus</i> essential oil (CKP-25-EO) into a chitosan nanoemulsion and efficacy assessment for inhibition of fungal inhabitation and aflatoxin B<sub>1</sub> (AFB<sub>1</sub>) contamination in <i>Syzygium cumini</i> seeds with emphasis on cellular and molecular mechanism of action. DLS, AFM, SEM, FTIR, and XRD analyses revealed the encapsulation of CKP-25-EO in chitosan with controlled delivery. The CKP-25-Ne displayed enhanced antifungal (0.08 µL/mL), antiaflatoxigenic (0.07 µL/mL), and antioxidant activities (IC<sub>50 DPPH</sub> = 6.94 µL/mL, IC<sub>50 ABTS</sub> = 5.40 µL/mL) in comparison to the free EO. Impediment in cellular ergosterol, methylglyoxal biosynthesis, and in silico molecular modeling of CKP-25-Ne validated the cellular and molecular mechanism of antifungal and antiaflatoxigenic activity. The CKP-25-Ne showed in situ efficacy for inhibition of lipid peroxidation and AFB<sub>1</sub> secretion in stored <i>S. cumini</i> seeds without altering the sensory profile. Moreover, the higher mammalian safety profile strengthens the application of CKP-25-Ne as a safe green nano-preservative against fungal association, and hazardous AFB<sub>1</sub> contamination in food, agriculture, and pharmaceutical industries.
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