This study aimed to evaluate the effect of two spacer materials (cotton pellet and polytetrafluoroethylene [PTFE]) on gap and void formation in the Cavit restoration used for endodontic temporization. Thirty-four extracted human single-rooted premolars were matched and allocated into two groups (n = 17/group) according to the access cavity spacer (cotton pellet or PTFE). Standardized endodontic access cavities were prepared in all the teeth. Subsequently, the teeth were scanned by micro-computed tomography (micro-CT) to determine the volume of the access cavities. The teeth were then temporized by placing either cotton pellet or PTFE as spacer materials on the canal orifices, followed by the Cavit restoration. Following the temporization procedure, the teeth were subjected to a second micro-CT scan to determine the percentage volume of gaps and voids along the margins and within the Cavit restoration, respectively. Statistical analysis was performed using the Shapiro–Wilk and Wilcoxon signed-rank tests with a 5% significance level. The PTFE spacer was associated with significantly less gap formation between the Cavit restoration and the access cavity walls (P < 0.05) compared with the cotton pellet. No difference existed in void formation between the groups (P > 0.05). These findings indicate that the spacer material placed under the Cavit restoration can influence the quality of overlying restoration. PTFE was associated with less gap formation and, therefore, performed better than the cotton pellet as a spacer material beneath the Cavit restoration during endodontic treatment.