Biofilm formation on surfaces is an ancient and integral strategy for bacterial survival. Billions of years of adaptation provide microbes with the ability to colonize any surface, including those used in orthopaedic surgery. Although remarkable progress has been made in the treatment of orthopaedic diseases with implanted prostheses, infection rates remain between 1% and 2%, and are higher for revision surgeries. The chronic nature of implant infections, their nonresponsiveness to antibiotics, and their frequent culture negativity can be explained by the biofilm paradigm of infectious disease. However, the role of biofilms in orthopaedic implant infections and aseptic loosening is controversial. To address these issues, we developed molecular diagnostic and confocal imaging techniques to identify and characterize biofilms associated with infected implants. We designed PCR and reverse transcription (RT)-PCR-based assays that can be used to detect bacterial infections associated with culture-negative joint effusions that distinguish between physiologically active Staphylococcus aureus and Staphylococcus epidermidis. Using clinical isolates of Pseudomonas aeruginosa, we constructed a series of reporter strains expressing colored fluorescent proteins to observe biofilms growing on 316L stainless steel and titanium orthopaedic screws. Three-dimensional structures of Pseudomonas aeruginosa and staphylococci biofilms growing on the screws were documented using confocal microscopy. The application of these tools for clinical diagnosis and biofilm research in animal and in vitro models is discussed.