The accurate preoperative diagnosis of periprosthetic joint infection (PJI) of total knee arthroplasty (TKA) can be difficult despite the use of a combination of serum and synovial markers. In such inconclusive cases, incisional open biopsy might be considered. This study investigates the usefulness of biopsies in patients with inconclusive diagnostic findings. We retrospectively identified 63 patients who underwent incisional biopsy for chronic PJI in the operation theatre following TKA revision between 2010 and 2018 after inconclusive preoperative diagnostics for PJI. In all cases, 5 independent biopsies were taken. Results from open biopsy for PJI were analyzed for diagnostic accuracy using the intraoperative results from following revision surgery as gold standard. 27 patients (43%) had a positive culture taken during biopsy. 15 cases (24%) met the diagnostic criteria for a chronic PJI. Most common organisms were Coagulase-negative staphylococci (67%) and 'Cutibacterium acnes' (30%). Compared to the findings during revision surgery, biopsies showed a sensitivity of 47% and a specificity of 77% for PJI. Open incisional biopsy following inconclusive serum- and synovial diagnostics for low grade PJI may be considered for identification of microorganisms. Due to its low sensitivity and moderate specificity found in the present cohort, microbiological analysis should be combined with additional diagnostic markers and histological investigation.