Bone allografts retrieved from multi-organ donors can be decontaminated with minimally aggressive methods. Therefore, we evaluated the efficacy of antibiotics and antiseptics in the decontamination of bone fragments actively contaminated with coagulase-negative staphylococci. Gentamicin (512/1,024 microg/mL), rifampicin (400/1,000 microg/mL), chlorhexidine in alcohol and chlorhexidine soap were tested with different contact times and temperatures and a delay in starting decontamination. Gentamicin-susceptible strains dried on bone could be removed by gentamicin 512 microg/mL after 19 h of contact, while strains not dried on bone could be eliminated by soaking bone for 60 min in gentamicin 512 microg/mL. Rifampicin-susceptible strains could be eliminated by soaking bone for 60 min in rifampicin 1,000 microg/mL. In none of the experimental conditions could gentamicin/rifampicin-resistant staphylococci be eliminated. Antiseptics could not eliminate staphylococci from bone. Different antibiotics need different protocols in order to decontaminate bone allografts.