The aim of this study was to evaluate the existence of preoperative dentinal defects among differently preserved dentoalveolar bone-blocks (frozen vs. embalmed) and to investigate the effect of varying apical forces (low: <4 N, high: 4–8 N) during root canal preparation on microcrack formation using micro-computed tomography (micro-CT). Thirteen embalmed and seven frozen bone-blocks containing 1–3 single rooted teeth were collected. The teeth were evenly divided into three groups (n = 10): FLow (frozen, <4 N), ELow (embalmed, <4 N), EHigh (embalmed, 4–8 N). After working length determination all specimens were scanned preoperatively. Root canal preparation was performed using nickel-titanium instruments sizes 25/.06 and 40/.06 (F6 SkyTaper; Komet, Lemgo, Germany). A postoperative scan was performed and image stacks were co-registered. All cross-sectional images were screened to identify the presence of dentinal defects. The results were expressed as the percentage of teeth/slices presenting dentinal defects. The statistical analyses were performed with Kruskal-Wallis-Test and Mann-Whitney-U-Test (α = 5%). Embalmed specimens presented a significantly higher percentage of slices with preoperative microcracks (p<0.05) than frozen specimens. No significant difference between groups was observed regarding the induction of microcracks (p>0.05). Root canal preparation does not induce microcracks in dentoalveolar bone-blocks from donors of old age, irrespective of the preservation method and the apically directed forces.