Fusarium head blight (FHB) is a widely occurring plant disease, which is caused by fungi in the genus Fusarium. FHB leads to mycotoxin accumulation on grain, which causes food safety risk and economic loss. In addition to chemical treatments, biological strategies, like application of lactic acid bacteria (LAB), could be useful in preventing and/or eradicating mycotoxigenic Fusarium growth and mycotoxin production.After comparision of the anti-Fusarium activities by a microdilution assay against Fusarium graminearum 08/RG/BF/51, Lactobacillus rhamnosus VT1 was found to have the highest anti-Fusarium activity. Response surface methodology (RSM) was employed to optimize the incubation conditions for the production of cell-free Lactobacillus culture supernatant (CFLCS) from the strain. The best combination included 34¨¬C, 55 hours, and shaking at 170 rpm for production of CFLCS from L. rhamnosus VT1. Under these incubation conditions, a 10% cell-free culture of Lactobacillus rhamnosus VT1 inhibited 83.7% of the Fusarium growth on microplate. MIC value of the CFLCS with a 10<super>4</super> conidia /well inoculum concentration is 18%.To identify the mechanisms of anti-Fusarium activity, a stepwise regression, with ¥á to enter = 0.15 and ¥á to remove = 0.15, was performed to analyze the data of the RSM design. It was indicated that pH, total acidity, and 3-phenyllactic acid were the most important factors and could be used to explain 39.2% variation of the anti-Fusarium activity. In addition, proteinaceous compounds might be important due to the possible synergistic effect in the CFLCS. CFLCS applied directly to grain not only prevented Fusarium growth, but also changed mycotoxin accumulation. Fusarium growth was inhibited completely by a 50% concentration (V/V) of the CFLCS applied on rice media after 14 days incubation, and almost no mycotoxins were detected. Concentrations of 15%, 30% and 50% of CFLCS as steeping water inhibited Fusarium growth and mycotoxin accumulation on barley in the malting process. Almost no mycotoxins were detected in the samples treated by 50% CFLCS. However, the germination ability of the barley samples was inhibited. In general, the CFLCS showed potential effective anti-Fusarium activity. However, the strategies of application of the CFLCS on grain should be further investigated.