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Aspergillus flavus is a major fungal phytopathogen and an opportunistic pathogen to humans and livestock. The fungus produces immunosuppressive and carcinogenic aflatoxins that acts as a burden in food and feed industries. Phylogenetic analysis indicated that the cell wall protein, A. flavus mannoprotein 1 (AFLMP1), is exclusively present in Aspergillus section Flavi such as A. flavus and A. parasiticus. This makes AFLMP1 an excellent candidate for siRNA-based control of aflatoxigenic fungi in farms and processing units, and fungal therapy in hospitals. Here and for the first time, mode of action of a chemically synthesized RNA interference (siRNA) was investigated for the control of AFLMP1 synthesis. The efficacy of direct uptake of different concentration of siRNA on spore germination of A. flavus was monitored via Opera High Content Screening confocal microscope. siRNA caused growth inhibition at lower concentrations (0.65 nM) and germination failure (more than 90%) at higher concentrations (5 nM), most likely by interfering in mannoprotein biosynthesis. It is assumed that siRNA technology can be implemented as a promising suppressive agent in treatment of target genes for inactivation. It can be considered as an intervention in food/feed industries to control the development and reproduction of fungi to keep the fungal population below hazard critical points.