Objective: The aim of this study was to simulate reusing an opened package of bone substitute (BS) under typical clinical conditions, by analyzing bacterial growth in samples of resealed packages of alloplast and xenograft BS following different time periods up to a week. Materials and Methods: The experiment was performed in 2 stages: dry stage and wet stage. In the dry stage, BS samples were exposed to air for 15 minutes and then were sealed again in their original package. After several “aging” periods [no aging (t0), 24 hours (t1), and 1 week (t7)], BS samples were plated onto brain heart infusion agar and were incubated at 37 °C for 24 hours under aerobic conditions. Bacterial colonies were enumerated and colony forming units per 1 mL was calculated. In the wet stage, the experiment was repeated but BSs were left to age in a brain heart infusion broth to simulate a more supporting humid environment to encourage bacterial growth. Results: In the dry stage of the experiment, no bacterial growth was detected in both xenograft and alloplast samples (colony forming units/mL=0), in any of the time points (t0, t1, t7). In the wet stage, average bacterial growth was 0 for both groups in t0. In t1: 1.14 × 104±4.16 × 103 for the xenograft and 7.73 × 105± 9.99 × 105 for the alloplast. In t7: 1.64 × 109±1.82 × 108 in the xenograft compared with 8.86 × 108±4.00 × 108 for the alloplast. Analysis of variance with repeated measures showed a significant difference in comparison of the 2 BSs for each time group (t1 and t7) (P<0.005). Conclusion: Under dry conditions, in a resealed package, there was no bacterial growth for up to 1 week for both BSs. Once the BSs are introduced to a humid environment, which supports bacterial growth, the xenograft BS allowed more rapid bacterial growth, compared with the alloplastic BS.