Staphylococci and enterococci account for most deep infections associated with bone-anchored percutaneous implants for amputation treatment. Implant-associated infections are difficult to treat; therefore, it is important to investigate if these infections have a biofilm origin and to determine the biofilm antimicrobial susceptibility to improve treatment strategies. The aims were: (i) to test a novel combination of the Calgary biofilm device and a custom-made susceptibility MIC plate (Sensititre ), (ii) to determine the biofilm formation and antimicrobial resistance in clinical isolates causing implant-associated osteomyelitis, and (iii) to describe the associated clinical outcome. Enterococci and staphylococci were characterized by microtitre plate assay, Congo Red Agar plate test, and PCR. Biofilm susceptibility to 10 antimicrobials and its relationship to treatment outcomes were determined. The majority of the strains produced biofilm in vitro showing inter- and intraspecies differences. Biofilms showed a significantly increased antimicrobial resistance compared with their planktonic counterparts. Slime-producing strains tolerated significantly higher antimicrobial concentrations compared with non-producers. All seven staphylococcal strains carried ica genes, but two did not produce slime. The degree of biofilm formation and up-regulated antibiotic resistance may translate into a variable risk of treatment failure. This new method set-up allows for the reproducible determination of minimum biofilm eradication concentration of antimicrobial agents, which may guide future antimicrobial treatment decisions in orthopaedic implant-associated infection. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2630-2640, 2017.