<p><i>Fusarium graminearum</i> infects barley easily, causing Fusarium head blight and the subsequent production of mycotoxin deoxynivalenol, which poses significant health risks to both humans and animals. Grains contaminated with deoxynivalenol can only be discarded, resulting in enormous waste. In previous study, carvacrol-loaded chitosan nanoparticles that inhibited the growth of <i>F. graminearum</i> were developed to save the economic losses caused by deoxynivalenol contamination in barley malt. However, its antifungal efficacy and mechanism of action remain unclear, and the present study therefore seeks to elucidate both. The results demonstrated that these nanoparticles effectively inhibited spore germination and germ tube elongation of <i>F. graminearum</i> when the concentrations exceeding 200&#xa0;µg·mL^− 1, thereby impeding its mycelial growth. Furthermore, the measurement of relative electrical conductivity revealed that treatment with carvacrol-loaded chitosan nanoparticles significantly increased the relative electrical conductivity of <i>F. graminearum</i> cells, which was attributed to severe disruption of cell membrane integrity and consequent leakage of intracellular proteins and nucleic acids. In the barley storage experiment, the content of ergosterol and deoxynivalenol in rehydration spray-treated barley malt was 2.2 times (with 200&#xa0;mg carvacrol-loaded chitosan nanoparticles addition) and 7.3 times (with 500&#xa0;mg carvacrol-loaded chitosan nanoparticles addition) higher than that of carvacrol-loaded chitosan nanoparticles treated barley malt. At the same time, they maintained the malt quality parameters (including moisture, extract content, saccharification time, etc.) without significant alteration. These findings carvacrol-loaded chitosan nanoparticles can serve as an environmentally friendly and highly effective antifungal agent, and their large-scale application will help promote global food security and sustainable agricultural development.</p>