ABSTRACT Osthole is a natural coumarin that exhibits wide biological and pharmacological activities such as neuroprotective, osteogenic, immunomodulation, antitumor, and anti-inflammatory effects. In this study, we investigated the antifungal effects of osthole in vitro . A checkerboard microdilution assay showed that osthole has significant synergistic effect with fluconazole against fluconazole-resistant Candida albicans . Similar results were obtained from a growth curve assay. Meanwhile, XTT reduction assay demonstrated the synergism of fluconazole and osthole against C. albicans biofilm formation. Microarray results showed that the expression of genes involved in the oxidation-reduction process, energy metabolism, and transportation changed significantly after the combined treatment with fluconazole and osthole, and further results showed that endogenous reactive oxygen species (ROS) was significantly increased in the combination group. In conclusion, these results demonstrate the synergism of fluconazole and osthole against fluconazole-resistant C. albicans and indicate that endogenous ROS augmentation might contribute to the synergism of fluconazole and osthole.