The growing trends of emergence of antifungal-resistant Candida strains has recently been inspired the researchers to design new antifungal agents with novel mechanisms of action. Glabridin is an originally natural substrate with multiple biological activities which propose it as a novel anticancer, antimicrobial and antifungal agent. In the present study, the antifungal effect of glabridin against Candida glabrata isolates and its possible mechanism of action were investigated. The minimum inhibitory concentrations (MIC) for glabridin against fluconazole-resistant and fluconazole-SDD strains of C. glabrata were investigated using the Clinical and laboratory standards institute document M27-A3 and M27-S4 as a guideline. Possible alternations in the expression of two critical genes involved in yeast apoptosis, MCA1 and NUC1, were assayed by real-time PCR. DNA damage and chromatin condensation was investigated using DAPI staining. Although glabridin led to a significant decrease in MICs against fluconazole-resistant C. glabrata (MIC₅₀: 8μg/mL), no significant decreased was shown for fluconazole-SDD strains. Therefore, a distinct azole-independent mechanism could be responsible for the inhibitory activity of glabridin. Overexpression of MCA1 and NUC1 genes in addition to DNA damage and chromatin condensation suggesting the involvement of apoptosis signaling in C. glabrata stains exposed to glabridin. This study suggests that glabridin might be considered as a novel naturally originated agent to fight against fluconazole-resistance C. glabrata strains.