Sprassis crispa was cultivated using soybean curd whey, and its antimicrobial activities were examined against those of eight microorganisms that were foodborne pathogens or food-poisoning bacteria. The culture broth of soybean curd whey was superior in mycelium content (17.76 g/L) to that of the defined culture broth, and the β-glucan content was about 10.64 percent (w/w). The antimicrobial activities of the culture broth were confirmed against those of B. cereus, St. aureus, L. monocytogenes and S. typhimurium using the paper disk method. The antimicrobial activity was also maintained after the heat treatment and alcalase treatment. The filtrate with less than 3 kDa M.W. also showed the antimicrobial activity against four strains: B. cereus, St. aureus, L. monocytogenes and S. typhimurium. The minimum inhibitory concentration (MIC) was about 1.26 mg/mL in the B. cereus and 12.6 mg/mL in the St. aureus and L. monocytogenes. The S. typhimurium showed a MIC of 62.8 mg/mL. Thus, the culture of Sparassis crispa using soybean curd whey provides a thermally stable antimicrobial agent that can be used as a natural preservative in the biofood industry.