Introduction: Fungal infections caused by Candida glabrata have significantly increased in recent years, particularly in hospital settings, posing a serious public health issue due to their growing resistance to conventional antifungals, especially azoles. In response, combining conventional antifungal agents with natural compounds like essential oils represents an innovative and promising therapeutic strategy. Materials and Methods: This study aimed to evaluate the antifungal activity of fluconazole and Origanum vulgare essential oil, both individually and in combination, against a clinical strain of Candida glabrata isolated from a urinary sample at the Hassan II Regional Hospital in Agadir. The study employed two complementary approaches: the disk diffusion method and liquid microdilution, allowing for the determination of inhibition zones and minimum inhibitory concentrations (MIC) of each agent. Results: The results from the diffusion method indicated that fluconazole exhibited moderate activity with an inhibition zone of 29.5 ± 0.5 mm and an MIC of 8 µg/mL, while O. vulgare essential oil demonstrated marked antifungal activity, with an inhibition zone of 62 ± 0.5 mm and an MIC of 0.0435 mg/mL. The combination of both agents resulted in an increased inhibition zone and a significant reduction in their respective MICs, with a Fractional Inhibitory Concentration Index (FICI) of 0.5, indicating a synergistic effect between fluconazole and the essential oil. This combination resulted in a fourfold enhancement of fluconazole's antifungal activity. Conclusion: In conclusion, these findings suggest that Origanum vulgare essential oil could enhance the effectiveness of fluconazole against Candida glabrata, opening interesting perspectives for the development of new combined antifungal approaches, particularly in the context of resistance to conventional treatments.