Fungal prosthetic joint infections (PJI) and reports of their clinical investigation are rare. In addition, there has been little evidence regarding the outcome of the two-stage exchange protocol for the treatment of fungal PJI. In order to investigate the risk factors and clinical, microbiological and pathological features of fungal PJIs, as well as the effects of the two-stage exchange protocol on their outcome, the present study analyzed eight retrospective fungal PJI cases, involving four cases affecting the hips and four affecting the knees, between May 2000 and March 2012. In all cases, a cemented spacer saturated with antimicrobials was used during the two-stage exchange protocol, and systematic antifungal agents were administrated during the interim period. The average follow-up duration was 4.4 years. Of the eight cases, six had undergone additional surgery on the infected joint prior to infection with the fungus. Following histological analyses, it was determined that the average number of polymorphonuclear cells in the three patients infected with a fungus was only <5/high power field (HPF; magnification, ×400), and that of the five patients with a hybrid infection was >5/HPF. The average Harris Hip scores or Hospital for Special Surgery knee scores were 43.6 preoperatively and 86 at the last follow-up. The two-stage exchange protocol was performed eight times in seven cases, with a failure rate of 12.5%. The remaining case was successfully treated by resection arthroplasty. The average duration of antifungal agent administration during the interim period in five of the eight cases was 1.5 months. For three of the patients, the duration of antifungal agent administration was prolonged until the c-reactive protein levels were decreased to normal. The average duration of spacer implantation into the joint was 4.3 months. The results of the present study suggested that undergoing surgery on a prosthetic joint may be a potential risk factor for the development of fungal PJI. In addition, infiltration of polymorphonuclear leukocytes into the site of the infection may not occur at the same rate as bacterial PJI. Therefore, a two-stage exchange protocol with implantation of a cement spacer saturated with antimicrobials may be considered an effective therapeutic strategy for the treatment of fungal PJI.