Bacterial infections are serious complications after orthopaedic implant surgery. Staphylococci, with Staphylococcus epidermidis as a leading species, are the prevalent and most important species involved in orthopaedic implant-related infections. The biofilm mode of growth of these bacteria on an implant surface protects the organisms from the host’s immune system and from antibiotic therapy. Therapeutic agents that disintegrate the biofilm matrix would release planktonic cells into the environment and therefore allow antibiotics to eliminate the bacteria. An addition of a biofilm-degrading agent to a solution used for washing–draining procedures of infected orthopaedic implants would greatly improve the efficiency of the procedure and thus help to avoid the removal of the implant. We have previously shown that the extracellular staphylococcal matrix consists of a poly-N-acetylglucosamine (PNAG), extracellular teichoic acids (TAs) and protein components. In this study, we accessed the sensitivity of pre-formed biofilms of five clinical staphylococcal strains associated with orthopaedic prosthesis infections and with known compositions of the biofilm matrix to periodate, Pectinex Ultra SP, proteinase K, trypsin, pancreatin and dispersin B, an enzyme with a PNAG-hydrolysing activity. We also tested the effect of these agents on the purified carbohydrate components of staphylococcal biofilms, PNAG and TA. We found that the enzymatic detachment of staphylococcal biofilms depends on the nature of their constituents and varies between the clinical isolates. We suggest that a treatment with dispersin B followed by a protease (proteinase K or trypsin) could be capable to eradicate biofilms of a variety of staphylococcal strains on inert surfaces.