Fusarium mycotoxins represent the most food safety concern for the malting industry. The complete prevention of Fusarium mycotoxins in the grains by limiting toxigenic fungi in the field and during storage is not practical. The common way to control Fusarium mycotoxins in malting industry is to avoid infected grains. However, avoidance is not always possible. To ensure the quality and safety of food products, development of food-grade antifungal strategies that can be applied in food processing, would benefit growers and the food industry. Recently, plant-based essential oils (EOs) have received considerable attentions in the food industry due to broad-spectrum of antifungal activities and inhibitory effect against mycotoxin biosynthesis. However, direct application of EOs during the malting process is impractical. In this project, parameters that impact on the formation of EO-in-water nanoemulsions and functional properties including antifungal and mycotoxin inhibitory efficacy were evaluated in vitro. The proper-designed EO-in-water nanoemulsions were then applied in micro-malting process. Results indicated that physically stable EO-in-water nanoemulsions can be fabricated by incorporating either ≥75 wt% of corn oil or ≥50 wt% of medium chain triacylglycerol (MCT) into EO before homogenization and homogenized under optimized processing conditions (68.95 MPa and 2 passes). In general, the mycotoxin inhibitory efficacy of EO was enhanced considerably in nanoemulsion form than bulk oil. Among all selected five EOs, thyme and clove oil-in-water nanoemulsions had the greatest antifungal and mycotoxin inhibitory activities. In terms of emulsifiers, the antifungal activity was mainly dominated by EO rather than emulsifier to alter mycelial and spore cell membrane integrity. At last, clove oil-in-water nanoemulsions stabilized by three different emulsifiers (Tween 80, bovine serum albumin, quillaja saponins) were selected to apply in micro-malting process according to our germinative energy test of barley seeds. All clove oil-in-water nanoemulsions had the capability to inhibit fungal growth and DON production during the micro-malting process. Among the three emulsifiers, Tween 80-stablized clove oil nanoemulsion displayed largest reduction of mycotoxin and least flavor impact on the final malt. The overall project showed a great potential for utilization of EO-in-water nanoemulsion as antifungal agent and mycotoxin inhibitor in the food industry.