The emergence of resistantCandida albicanshas made clinical fluconazole (FLC) treatment difficult. Improving sensitivity to FLC is an effective way to treat resistant isolates. Berberine hydrochloride (BBH) is a commonly used traditional Chinese medicine with antimicrobial effects, especially in resistant isolates. We investigated the molecular mechanisms underlying BBH and FLC synergism on biofilm-positive FLC-resistantC. albicansinhibition. Checkerboard microdilution assays and time-kill assays showed a strong synergistic effect between BBH and FLC in resistantC. albicansisolates, causing a significant 32-512-fold reduction in minimum inhibitory concentrations. BBH combined with FLC inhibited intracellular FLC efflux due to key efflux pump geneCDR1downregulation, whereas FLC alone induced highCDR1transcription in resistant strains. Further, BBH + FLC inhibited yeast adhesion, morphological hyphae transformation, and biofilm formation by downregulating the hyphal-specific genesALS3,HWP1, andECE1. BBH caused cytoplasmic Ca(2+)influx, while FLC alone did not induce high intracellular Ca(2+)levels. The vacuolar calcium channel geneYVC1was upregulated, while the vacuolar calcium pump genePMC1was downregulated in the BBH + FLC and BBH alone groups. However, vacuolar calcium gene expression after FLC treatment was opposite in biofilm-positive FLC-resistantC. albicans, which might explain why BBH induces Ca(2+)influx. These results demonstrate that BBH + FLC exerts synergistic effects to increase FLC sensitivity by regulating multiple targets in FLC-resistantC. albicans. These findings further show that traditional Chinese medicines have multi-target antimicrobial effects that may inhibit drug-resistant strains. This study also found that the vacuolar calcium regulation genesYVC1andPMC1are key BBH + FLC targets which increase cytoplasmic Ca(2+)in resistant isolates, which might be critical for reversing biofilm-positive FLC-resistantC. albicans.