The potential of antifungal agents used as antimicrobial lock therapy (ALT) for the conservative management of catheter-related candidemia has not been fully defined. We sought to determine the antifungal effect of high-dose doxycycline (DOX), alone or in combination with standard concentrations of amphotericin B (AMB), caspofungin (CAS) or fluconazole (FLC), against biofilms formed by Candida albicans in vitro. DOX alone (at 2048 microg/mL and 1024 microg/mL) demonstrated up to an 85% reduction of the metabolic activity of the C. albicans biofilm. Regardless of the concentration tested, FLC alone showed minimal activity (mean 22.9% reduction) against the C. albicans biofilm. When DOX 2048 microg/mL was used in combination with FLC, antifungal activity also increased up to 85%, suggesting an additive effect. DOX 128 microg/mL in combination with FLC demonstrated synergy (mean 58.3% reduction). The combination of DOX 2048 microg/mL or 512 microg/mL and AMB was superior to AMB alone at low concentrations (0.25-0.03125 microg/mL). However, DOX 128 microg/mL was antagonistic in combination with low concentrations of AMB. Maximal efficacy against the biofilm was observed with CAS at 8-0.25 microg/mL compared with FLC and AMB alone. A paradoxical effect (PE) occurred with CAS at 16 microg/mL, which showed a marked reduction in antifungal activity compared with lower concentrations of CAS. CAS at 16 microg/mL in combination with either DOX 2048 microg/mL or 512 microg/mL resulted in attenuation of the PE. These findings suggest that a high-dose DOX-based ALT strategy in combination with traditional antifungal agents may be useful for the treatment of C. albicans biofilms.