Background Access cavity preparation is a crucial step in nonsurgical root canal treatment. Recent advancements in access cavity designs focus on preserving maximum tooth structure while ensuring sufficient access to canal orifices for effective cleaning and shaping, resulting in minimally invasive procedures. However, there is limited information on the impact of three-dimensional (3D)-guided access cavity preparation in molars. A literature review found no prior studies comparing the effects of various access cavity preparation techniques on apical transportation, untouched surfaces, and debris formation within the canal. Objective The objective of this study is to compare and evaluate the effects of three different access cavity techniques on apical transportation, untouched surfaces, and debris formation within the root canal. Material and methods Thirty extracted permanent mandibular first molars were selected and randomly assigned to three groups for this study Group I received 3D-printed static guided cavity preparation, Group II underwent conservative access cavity preparation, and Group III was subjected to traditional access cavity (TAC) preparation. The mesial canals in all samples were cleaned and shaped using TruNatomy files. Preoperative and postoperative micro-CT imaging was performed on each sample to assess the effects of the different access cavity preparation techniques on apical transportation, untouched surfaces, and debris formation within the root canal. Results The study found that Group I, which used 3D-printed static guided cavity preparation, exhibited significantly less apical transportation compared to Groups II and III, with mean differences of -0.1677 and -0.2079, respectively. Debris accumulation was similar across all groups, with mean values of 0.928 ± 0.824 for Group I, 0.751 ± 0.495 for Group II, and 0.938 ± 0.681 for Group III, indicating no significant impact of cavity preparation type on debris levels. For untouched canal surfaces, Group III (TAC preparation) had the fewest untouched surfaces, with mean differences of 3.0380 and 3.9020 compared to Groups II and I, respectively. Conclusions While TAC preparation reduces substantial tooth structure, it shows higher instrumentation efficacy and better cleaning of the root canal system. However, in complex cases where tooth structure preservation is crucial, guided access cavity preparation provides an effective balance between structural conservation and adequate canal access. This approach offers a tailored solution, optimizing treatment outcomes based on the specific clinical scenario.