OBJECTIVE The authors' aim was to report the largest study on predictors of infection after cranioplasty and to assess the predictive value of intraoperative bone flap cultures before cryopreservation. METHODS They retrospectively examined all cranioplasties performed between March 2004 and November 2014. Throughout this study period, the standard protocol during initial craniectomy was to obtain a culture swab of the extracted autologous bone flap (ABF)—prior to its placement in cytostorage—to screen for microbial contamination. Two consecutive protocols were employed for the use and interpretation of the intraoperative swab culture results: A) From March 2004 through June 2013, any culture-positive ABF (+ABF) was discarded and a custom synthetic prosthesis was implanted at the time of cranioplasty. B) From July 2013 through November 2014, any ABF with a skin flora organism was not discarded. Instead, cryopreservation was maintained and the +ABF was reimplanted after a 10-minute soak in bacitracin irrigation as well as a 3-minute soak in betadine. RESULTS Over the 10.75-year period, 754 cranioplasty procedures were performed. The median time from craniectomy to cranioplasty was 123 days. Median follow-up after cranioplasty was 237 days for protocol A and 225 days for protocol B. The overall infection rate after cranioplasty was 6.6% (50 cases) occurring at a median postoperative Day 31. Staphylococcus spp. were involved as the causative organisms in 60% of cases. Culture swabs taken at the time of initial craniectomy were available for 640 ABFs as 114 ABFs were not salvageable. One hundred twenty-six (20%) were culture positive. Eighty-nine +ABFs occurred during protocol A and were discarded in favor of a synthetic prosthesis at the time of cranioplasty, whereas 37 +ABFs occurred under protocol B and were reimplanted at the time of cranioplasty. Cranioplasty material did not affect the postcranioplasty infection rate. There was no significant difference in the infection rate among sterile ABFs (7%), +ABFs (8%), and synthetic prostheses (5.5%; p = 0.425). All 3 +ABF infections under protocol B were caused by organisms that differed from those in the original intraoperative bone culture from the initial craniectomy. A cranioplasty procedure ≤ 14 days after initial craniectomy was the only significant predictor of postcranioplasty infection (p = 0.007, HR 3.62). CONCLUSIONS Cranioplasty procedures should be performed at least 14 days after initial craniectomy to minimize infection risk. Obtaining intraoperative bone cultures at the time of craniectomy in the absence of clinical infection should be discontinued as the culture results were not a useful predictor of postcranioplasty infection and led to the unnecessary use of synthetic prostheses and increased health care costs.