Biofilm formation is a serious problem that relatively often causes complications in orthopedic surgery. Biofilm-forming pathogens invade implanted foreign bodies and surrounding tissues. Such a condition, if not limited at the appropriate time, often requires reoperation. This can be partially prevented by selecting an appropriate prosthesis material that prevents the development of biofilm. There are many modern techniques available to detect the formed biofilm. By applying them we can identify and visualize biofilm-forming microorganisms. The most common etiological factors associated with biofilms in orthopedics are: Staphylococcus aureus, coagulase-negative Staphylococci (CoNS), and Enterococcus spp., whereas Gram-negative bacilli and Candida spp. also deserve attention. It seems crucial, for therapeutic success, to eradicate the microorganisms able to form biofilm after the implantation of endoprostheses. Planning the effective targeted antimicrobial treatment of postoperative infections requires accurate identification of the microorganism responsible for the complications of the procedure. The modern microbiological testing techniques described in this article show the diagnostic options that can be followed to enable the implementation of effective treatment.