Two-stage revision surgery is the current gold standard for treating prosthetic joint infections (PJI). Between the first and the second stage gentamicin-loaded (G) spacers are widely used but the rate of gentamicin resistant staphylococci is increasing. The potential benefit of vancomycin + gentamicin-loaded (V/G) spacers has not yet been evaluated. The aim of our study was to compare the microbiological eradication and infection control rates in PJI treated with G- or V/G-spacers. 147 PJIs treated in our institution were retrospectively reviewed. From 2003 to 2009 G-spacers (Tecres®) were used (group G) and from 2010 to 2013 V/G-spacers (Group V/G). Gender, age, body mass index (BMI), co-morbidities, ASA score, type of infection, microorganisms isolated in the first and second stages, time between stages, infection outcome at last visit were collected. The 2 main outcome variables were microbiological eradication in the second stage (≤1 positive culture out of 6) and infection control after the second stage. Univariate and multivariate analysis were performed using SPSS®. There were 83 patients in group G and 63 in group V/G. The mean (SD) age was 71.5 (10.3) years and 54% were female. Groups were similar in gender, age, BMI, ASA score, time with spacer, microorganism isolated in the first stage, or type of infection (acute or chronic) (p>0.05). The presence of ≥2 positive cultures in the second stage was significantly higher in group G (23.2%) than in group V/G (6.7%, P<0.05). Logistic regression model identified polymicrobial infections (OR: 4.26, CI95%: 1.44–12.64) and the use of G-spacers (OR: 5.88, CI95%: 1.60–21.74) as independent predictors of failure in microbiological eradication. The global rate of infection control was 75% after a mean (SD) follow-up of 56 (32) months. Infection control was higher in chronic than acute PJI (83.6% vs 59.6%, P<0.05), when cultures during second stage were negative (81.5%) vs positive (61%, P<0.05), and there was a trend towards a higher control rate when V/G-spacers (82%) vs G-spacers (69.5%) were used (P=0.09). Multivariate analysis identified chronic PJI (OR: 5.43, CI95%: 2.20–13.51) and, at the limit of significance, the use of V/G spacers (OR: 2.36, CI95%: 0.97–5.71) as predictors of infection control. Vancomycin loaded spacers were significantly associated with a higher microbiological eradication and there was a trend towards a higher infection control than gentamicin loaded spacers.