Objective: The aim of this paper was to study the outcome of patients who underwent cranioplasty with cryopreserved autologous bone after decompressive craniectomy. Methods: Data from 74 patients were retrospectively analyzed. They were divided into groups according to the storage time and the age at cranioplasty. To assess its predictive potential for complication, factors were related to successive stages (preoperative, craniectomy, tissue processing, cranioplasty, and postoperative). Cooling and warming rates applied on bone flap were calculated. The ability to inhibit microbial growth was determined exposing bone fragments to a panel of microorganisms. The concentration of antibiotics eluted from the bone was also determined. A bone explant culture method was used to detect living cells in the thawed cranial bone. Results: Hydrocephalus was significantly more frequent in pediatric patients (26.7%) than in adults (5.1%). The overall rate of bone flap resorption was 21.6% (43.7% of them requiring reoperation). Surgical site infection after cranioplasty was detected in 6.8% of patients. There was no correlation between infection as postoperative complication and previous microbiological positive culture during processing. The etiology of craniectomy did not influence the risk of bone flap contamination. Vancomycin was the only antibiotic detected in the supernatant where the bone was incubated. Outgrowth from bone explants was observed in 36.8% of thawed skulls. An early start of bone flap processing at the tissue bank had a positive effect on cell viability. Conclusion: The outcome after autologous cranioplasty is a multifactorial process, which is modulated by patient-, surgery-, and bone-related factors.