Microbiological cultures are moderately sensitive for diagnosing prosthetic joint infection (PJI). This study was conducted to determine whether amplification-based DNA methods applied on intraoperative samples could enhance PJI diagnosis compared with culture alone in routine surgical practice. Revision arthroplasty was performed for suspected PJI (n = 41) and osteoarthrosis control (n = 28) patients, and a diagnosis of PJI was confirmed in 34 patients. Amplification by polymerase chain reaction was performed on both 16S ribosomal DNA universal target genes and femA Staphylococcus-specific target genes. Species identification was achieved through amplicon sequencing. Amplification of the femA gene led to subsequent testing for methicillin resistance by amplification of the mecA gene. Microbiological and molecular assays identified a causative organism in 22 of 34 patients (64.7%) and in 31 of 34 patients (91.2%), respectively. In 18 of the 22 culture-positive patients, molecular and microbiological results were concordant for bacterial genus, species, and/or methicillin resistance. Bacterial agents were identified only by molecular methods in nine PJI patients, including seven who were receiving antibiotics at the time of surgery and one with recent but not concomitant antibiotherapy. DNA-based methods were found to effectively complement microbiological methods, without interfering with existing procedures for sample collection, for the identification of causative pathogens from intraoperative PJI samples, especially in patients with recent or concomitant antibiotherapy.