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Background Periprosthetic joint infections (PJI) caused by pathogens, for which no biofilm-active antibiotics are available, are often referred to as difficult-to-treat (DTT). However, it is unclear whether the outcome of DTT PJI is worse than those of non-DTT PJI. We evaluated the outcome of DTT and non-DTT PJI in a prospective cohort treated with a two-stage exchange according to a standardized algorithm. Methods Patients with hip and knee PJI from 2013 to 2015 were prospectively included and followed up for ≥ 2 years. DTT PJI was defined as growth of microorganism(s) resistant to all available biofilm-active antibiotics. The Kaplan–Meier survival analysis was used to compare the probability of infection-free survival between DTT and non-DTT PJI and the 95% confidence interval (95% CI) was calculated. Results Among 163 PJI, 30 (18.4%) were classified as DTT and 133 (81.6%) as non-DTT. At a mean follow-up of 33 months (range 24–48 months), the overall treatment success was 82.8%. The infection-free survival rate at 2 years was 80% (95% CI 61–90%) for DTT PJI and 84% (95% CI 76–89%) for non-DTT PJI (p = 0.61). The following mean values were longer in DTT PJI than in non-DTT PJI: hospital stay (45 vs. 28 days; p < 0.001), prosthesis-free interval (89 vs. 58 days; p < 0.001) and duration of antimicrobial treatment (151 vs. 117 days; p = 0.003). Conclusions The outcome of DTT and non-DTT PJI was similar (80–84%), however, at the cost of longer hospital stay, longer prosthesis-free interval and longer antimicrobial treatment. It remains unclear whether patients undergoing two-stage exchange with a long interval need biofilm-active antibiotics. Further studies need to evaluate the outcome in patients treated with biofilm-active antibiotics undergoing short vs. long interval.