Combination therapy can be used for the treatment of fungal infections, especially for those caused by antifungal-resistant fungi. In the present study, in vitro interactions and mechanisms between fluconazole and minocycline against Candida albicans were evaluated. The nature of the interactions determined by spectrophotometric method in a checkerboard assay was interpreted using nonparametric models of fractional inhibitory concentration index (FICI) and percentages of growth difference (ΔE). In the mechanism study, we evaluated the potential activity of minocycline on fluconazole penetrating the C. albicans biofilm. Furthermore, the effect of fluconazole and minocycline alone and in combination on the cellular calcium balance, as well as on the uptake and efflux of fluconazole were evaluated. It was found that fluconazole can work synergistically with minocycline against fluconazole-resistant C. albicans; the minimum inhibitory concentration of fluconazole decreased from 512 to 2 microgmL(-1) when fluconazole and minocycline were given in combination, with an FICI of 0.035 and 0.064 and high-percentage synergistic interactions of 1250% and 988% for the two resistant strains. The mechanism of action was suggested to be the enhancement of minocycline on fluconazole penetrating biofilm, and inducing the intracellular calcium release, instead of impacting on the uptake and efflux of fluconazole. Our results suggest that the combination of fluconazole and minocycline can reduce the fluconazole resistance of C. albicans in vitro.