Abstract—Binary biofilms containing gram-positive bacteria Micrococcus thailandicus HB or Kocuria rhizophila strain 4A-2G and gram-negative bacteria Escherichia coli strain K-12 or strain ET12567 pRAG56 were obtained. Strain E. coli ET12567 contained a plasmid encoding aminoglycoside phosphotransferase, the enzyme which inactivates kanamycin. A new kinetic approach was developed for selective detection of metabolically active gram-positive bacteria in binary biofilms with E. coli, based on the differences in reduction rate of the electron acceptor, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). It was found that the studied E. coli strains did not exert a protective effect upon the action of antibiotics vancomycin and oxacillin on gram-positive bacteria in the binary biofilms under formation. However, a protective effect was found in the case of preformed binary biofilms: the growth of gram-positive bacteria in binary biofilms was observed at the concentrations of antibiotics oxacillin and kanamycin, which almost completely suppressed growth in the mono-species biofilms of these gram-positive bacteria. The presence of a protective effect was confirmed by several methods (dynamics of MTT reduction, densitometric evaluation of biofilm staining, measurement of the optical density of MTT extracts, and CFU enumeration).