In ancient China, Ganoderma lucidum was believed to be a medical fungus that could increase lifespan. Recently, pharmacologic studies have found that polysaccharide peptides and triterpenoids extracted from Ganoderma lucidum have various physiological effects as active compounds. However, the effects of spore oil isolated from Ganoderma lucidum remains unknown. In this study, the biological effects of Ganoderma lucidum spore oil (GLSO) were evaluated using a Drosophila melanogaster model. Compared with untreated groups, groups treated with GLSO had significantly longer average and maximum lifespan in both normal conditions and under oxidative stress. The activities of various antioxidant enzymes were measured to determine the antioxidant effect of GLSO. GLSO treatment markedly enhanced total superoxide dismutase (SOD) and catalase (CAT) activity and decreased levels of malondialdehyde (MDA). Further, we found dose-dependent increases in the mRNA expression of Cu, Zn-SOD, Mn-SOD, and CAT in GLSO-treated groups. These results suggest that GLSO may effectively eliminate free radicals and extend lifespan in Drosophila. Future work should investigate the value of GLSO as a functional food for the prevention of aging in larger animal models.