In the last decades, it has been shown that biofilm-associated infections in most cases are caused by rather two or even more pathogens than by single microorganisms. Due to intermicrobial interactions in mixed communities, bacteria change their gene expression profile, in turn leading to alterations in the biofilm structure and properties, as well as susceptibility to antimicrobials. Here, we report the alterations of antimicrobials efficiency in mixed biofilms ofStaphylococcus aureus–Klebsiella pneumoniaein comparison with mono-species biofilms of each counterpart and discuss possible mechanisms of these alterations. In cell clumps detached from dual-species biofilms,S. aureusbecame insensitive to vancomycin, ampicillin, and ceftazidime compared to solelyS. aureuscell clumps. In turn, the increased efficiency of amikacin and ciprofloxacin against both bacteria could be observed, compared to mono-species biofilms of each counterpart. Scanning electron microscopy and confocal microscopy indicate the porous structure of the dual-species biofilm, and differential fluorescent staining revealed an increased number of polysaccharides in the matrix, in turn leading to more loose structure and thus apparently providing increased permeability of the dual-species biofilm to antimicrobials. The qRT-PCR showed thaticaoperon inS. aureusbecame repressed in mixed communities, and polysaccharides are produced mainly byK. pneumoniae. While the molecular trigger of these changes remains undiscovered, detailed knowledge of the alterations in antibiotic susceptibility to given drugs opens doors for treatment correction options forS. aureus–K. pneumoniaebiofilm-associated infections.