This study was performed in order to analyze the fibrinolytic, thrombin inhibitory, anti-oxidative, acetylcholinesteraseinhibitory, and immuno-enhancing activities of the water extract and solvent fractions isolated from Grifola frondosa. Fibrinolyticactivity was analyzed using the fibrin plate method, and thrombin inhibitory activity was assayed using the substrate H-D-Phe-pip-arg-pna. Anti-oxidative activity was estimated using the DPPH assay, and AChE inhibitory activity was measured using thespectrophotometric method. Immuno-enhancing activity was examined using the nitric oxide (NO) production in RAW 264.7macrophage cells. Cell viability was determined using the MTS assay. Fibrinolytic activities were the highest in water extract (1.55plasmin units/mL) followed by water fraction (0.85 plasmin units/mL). The thrombin inhibitory activities of the water and ethylacetate fractions were determined to be 76.43% and 72.59%, respectively. The acetylcholinesterase inhibitory activities ofchloroform and hexane fractions exhibited values of 95.14% and 94.74%, respectively. The butanol fraction showed the highestanti-oxidative activity at 94.47%. Anti-proliferating activity against Raw 264.7 cells showed no cytotoxicity. The production of NOin Raw 264.7 cells increased up to 2-fold by adding the water fraction compared to the untreated control. These results suggestthat Grifola frondosa may serve as a useful functional food for the enhancement of immune function and the prevention andtherapy of cardiovascular diseases.