The roots of Rosa multiflora Thunberg have been used in traditional oriental medicine as remedies for rheumatic arthralgia and scabies. In this study, the anti-fungal, anti-oxidant, and anti-inflammatory activities of a supercritical extract of Rosa multiflora root were investigated in vitro. To investigate the anti-oxidant and anti-inflammatory effects of the supercritical extract, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging activity, and the inhibition of nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells were examined, respectively. In addition, the anti-fungal activities of the extract were assessed. The results showed a concentration-dependent, increase in ABTS radical scavenging activity. The supercritical fluid extracts of Rosa multiflora root exhibited low toxicity to RAW 264.7 cells at 100 μg/mL the highest concentration tested. Cells stimulated with LPS produced more nitric oxide than normal control cells; however, cells treated with the supercritical fluid extract decreased this production in a concentration-dependent manner. Finally, the supercritical fluid extracts showed significant anti-fungal activity. These results suggest that extracts of the roots of Rosa multiflora might be used to develop potent anti-fungal, anti-oxidant, and anti-inflammatory agents, and may be useful as ingredients for related new functional cosmetic materials.