Abstract: OBJECTIVE To study the effect and potential action mechanisms of omadacycline combined with fluconazole on inhibition of drug-resistant Candida albicans through in vitro and in vivo experimental study. METHODS By means of the checkerboard microdilution method and a Galleria mellonella infection model, the synergistic antifungal activity of omadacycline combined with fluconazole against the drug-resistant C. albicans was determined in vitro and in vivo. RNA sequencing (RNA-seq) was employed to further explore the synergistic action mechanisms of the drug combination. RESULTS The in vitro experiment demonstrated that the combination of omadacycline (4 μg/ml) and fluconazole (0.25 to 1) μg/ml exhibited potent synergistic activity against the drug-resistant C. albicans, with a fractional inhibitory concentration index (FICI) ranging from 0.0161 to 0.0322. In the Galleria mellonella model infected with the drug-resistant C. albicans, the combination therapy (omadacycline 2 μg/larva + fluconazole 1 μg/larva) significantly increased the survival rate of the infected larvae (P< 0.05). Furthermore, RNA-seq and quantitative real-time PCR (qRT-PCR) analyses revealed that the synergistic inhibition mechanism of the drug combination on the drug-resistant C. albicans was associated with the disruption of integrity of fungal cell wall and the induction of intracellular ion homeostasis imbalance. CONCLUSION The combination of omadacycline and fluconazole can effectively inhibit the drug-resistant C. albicans both in vitro and in vivo, providing a promising novel therapeutic strategy for the treatment of the drug-resistant C. albicans infection.