© 2022, The Author(s) under exclusive licence to SICOT aisbl.Purpose: Re-operation after septic failure of a one-stage exchange for prosthetic joint infection (PJI) of the knee is a highly challenging procedure with concerns over residual bone stock, soft tissues, and stability. The associated changes in microbiology in cases of reinfection are still largely unknown. Methods: A comprehensive analysis was performed of all patients treated at our tertiary institution between 2001 and 2017 who developed reinfection after a one-stage exchange for PJI of the knee. Prerequisites for inclusion were a certain diagnosis of PJI according to the ICM criteria and a minimum follow-up of three years. Data on comorbidities, previous surgical interventions, microbiological findings at the time of the initial one-stage exchange and at the time of reinfection, detection methods, and antibiotic resistance patterns were retrospectively studied. Results: Sixty-six patients were identified that met the inclusion criteria. Reinfection occurred after a mean time interval of 27.7 months (SD ± 33.9, range 1–165). Ten types of bacteria were found that were not present before the one-stage exchange. The causative pathogen remained identical in 22 patients (33%) and additional microorganisms were detected in ten patients (15%). Half of the reinfections were however due to (a) completely different microorganism(s). A significant increase in the number of PJIs on the basis of high-virulent (23 vs 30, p = 0.017) and difficult-to-treat bacteria (13 vs 24, p = 0.035) was found. Conclusion: The present study provides a novel insight into the microbiological changes following septic failure after one-stage exchange for PJI of the knee. A higher prevalence of more difficult-to-treat bacteria might increase the complexity of subsequent procedures. Also, a longer follow-up of these patients than previously suggested seems in order.