Preliminary research found that mold growth was inhibited in fresh pasta containing flaxseed (Linum usitatissimum) flour (FF), which suggested that agents from flaxseed might help control mold. The objective of the current project was to assess the effect of cultivar, source, concentration, and heat treatment on antifungal activity of FF. A wet noodle model system was developed to determine the antifungal activities of FF. Sub-samples were taken to screen the antifungal activity. Noodle samples were stored in plastic bags at room temperature and checked daily for visual observation of mold growth. Spot inoculation method was used to further access the antifungal activity of FF towards specific fungi species. Penicillium chrysogenum, Aspergillus flavus, Fusarium graminearum and single spores isolated from the most prevalent molds in molded noodle were used as test microorganisms. Flaxseed flour from two cultivars collected from four seed companies located in Minnesota and North Dakota were used to determine the effect of cultivar and sources. Results showed that FF delayed the mold growth in the noodle system. Flour from both yellow ‘Omega’ and brown flaxseed had antifungal activity in the model noodle system regardless of flaxseed source, although difference in the degree of mold inhibition was found among FFs due to flaxseed source and cultivar. A concentration of 9% FF or higher was found to inhibit the growth of Noodle isolate and P. chrysogenum. In addition, 15% FF addition had the same effect as 0.2% propionic acid against the growth of P. chrysogenum. Flaxseed flour heated to 121°C for 15 min did not inhibit mold, demonstrating that the antifungal agent in flaxseed flour was heat labile.