Biofilms colonize medical devices and are often recalcitrant to antibiotics. Inter-kingdom biofilms, when at least a bacterium and a fungus are co-isolated, increase the likelihood of therapeutic failures. In this work, a three-species in vitro biofilm model including
S. aureus
,
E. coli
and
C. albicans
was used to study the activity of the antibiotics moxifloxacin and meropenem, the antifungal caspofungin, and combinations of them against inter-kingdom biofilms. The culturable cells and total biomass were evaluated to determine the pharmacodynamic parameters of the drug response for the incubation with the drugs alone. The synergic or antagonistic effects (increased/decreased effects) of the combination of drugs were analysed with the highest single agent method. Biofilms were imaged in confocal microscopy after live/dead staining. The drugs had limited activity when used alone against single-, dual- or three-species biofilms. When used in combination, additive effects were observed against single- or dual-species biofilms, and increased effects (synergy) against biomass of three-species biofilms. In addition, the two antibiotics showed different patterns, moxifloxacin being more active when targeting
S. aureus
and meropenem when targeting
E. coli
. All these observations were confirmed by confocal microscopy images. Our findings highlight the interest in combining caspofungin with antibiotics against inter-kingdom biofilms.