Implantation of antibiotic-loaded beads is accepted as an efficient option for local antibiotic therapy in orthopedic-related infections. However, recent reports have emphasized the bacteria growth persistence on antibiotic-impregnated bone cement. Hence, the aim of this study was to elaborate if bacterial adherence and growth could be determined on explanted gentamicin- and gentamicin-vancomycin-loaded beads after infection eradication. 18 chains of antibiotic-loaded beads (11 gentamicin-, 7 gentamicin-vancomycin-loaded) were examined. Indications for primary beads implantation included postoperative infections after total hip or knee arthroplasty, rotator cuff reconstruction, chronic foot osteomyelitis, anterior cruciate ligament reconstruction and dorsal spondylodesis. Among the isolated organisms, Staphylococcus epidermidis, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) were the most frequent ones. In 4 cases (3 × S. epidermidis, 1 × MRSA) bacteria growth persistence could be determined on the beads. S. epidermidis-strains persisted only on gentamicin-loaded beads, MRSA could grow on gentamicin-vancomycin-impregnated cement. In one case, the emergence of a gentamicin-resistant S. epidermidis-strain could be observed despite preoperative susceptibility. Bacteria growth persistence on bone cement is a hazardous problem in the orthopedic surgery and should therefore be born in mind. Adherence to cement can lead to emergence of bacteria resistance despite preoperative antibiotic susceptibility and might result in clinical recurrence of infection.