A novel synergistic formulation (TML) based on the combination of thymol (T), methyl cinnamate (M), and linalool (L) has been prepared using the mixture design assay. Nanoencapsulation of developed formulation TML (Ne-TML) was prepared and characterised by SEM, XRD and FTIR. The Ne-TML was assessed for its antifungal and anti-aflatoxin B potential in vitro and in the food systems (Pennisetum glaucum L.), and also examined its effects on organoleptic properties. The Ne-TML cause complete inhibition of growth and AFB production at 0.3 μl/ml and 0.2 μl/ml. In-situ results revealed that Ne-TML exhibited maximum protection from fungal (75.40%) and aflatoxin B contamination (100%) at 0.3 μl/ml during six months of storage. The speculated antifungal mode of action of Ne-TML was related to the decrease in ergosterol content, membrane ions leakage, impairment in carbon-source utilization, mitochondrial functioning, anti-oxidative defence system (SOD, CAT, and GR) and Ver-1 gene of aflatoxin B biosynthesis.