Background: Ganoderma lucidum is a well-known fungus that has been widely used in traditional medicine around the world, especially in East Asia, due to its various health promotion properties. Recently, researchers have drawn attention to the biologically active compounds found in this fungus, and this fungus has become very popular due to its pharmaceutical properties. Objectives: The aim of this study was to investigate the antifungal properties of the Iranian strain of G. lucidum as a natural antifungal agent against harmful filamentous fungi common in the food industry. Methods: Three filamentous fungi, including Aspergillus flavus, Aspergillus ochraceus, and Fusarium graminearum, were used in this study for the antifungal evaluation of ethanolic, hydroalcoholic, and two aqueous extracts of G. lucidum with different concentrations by the broth microdilution method. Results: The results showed that only the ethanolic and hydroalcoholic extracts completely inhibited the growth of A. flavus at 2 and 3.5 mg/mL, respectively. Also, no antifungal activity was observed for the aqueous extract for all the three studied fungi. In addition, A. flavus was found to be more sensitive to G. lucidum extracts compared to the two other studied fungi. Conclusions: The ethanolic extract of G. lucidum was effective on A. flavus and can be used as a natural antifungal agent to prevent the growth of this harmful filamentous fungus.