The aim of the study was to explore the antifungal and aflatoxin B₁ inhibitory efficacy of nanoencapsulated antifungal formulation. Mixture design response surface methodology (RSM) was utilized to design the antifungal formulation (SBC 4:1:1) based on the combination of chemically characterized Ocimum sanctum (S), O. basilicum (B), and O. canum (C) against Aspergillus flavus. The SBC was incorporated inside the chitosan nanomatrix (Ne-SBC) using an ultrasonic probe (40 kHz) and interactions were confirmed by SEM, FTIR and XRD analysis. The results showed that the Ne-SBC possessed enhanced antifungal and aflatoxin B₁ inhibitory effect over the free form of SBC. The biochemical and in silico results indicate that the antifungal and aflatoxin B₁ inhibitory effect was related to perturbance in the plasma membrane function (ergosterol biosynthesis and membrane cation) mitochondrial membrane potential, C-sources utilization, antioxidant defense system, and the targeted gene products Erg 28, cytochrome c oxidase subunit Va, and Nor-1. In-situ observation revealed that Ne-SBC effectively protects the Avena sativa seeds from A. flavus and AFB₁ contamination and preserves its sensory profile. The findings suggest that the fabrication of SBC inside the chitosan nano-matrix has promising use in the food industries as an antifungal agent.

Keywords: Antifungal; Chitosan; In-vivo efficacy; Mode of action; Statistical modeling.