This case report describes a novel design for an anatomical guide used in endodontic microsurgery (EM) with the cortical bone window technique. A 60-year-old female patient presented with persistent pain and a radiographic periapical lesion associated with tooth #36 (i.e., lower left first molar). Following a diagnosis of symptomatic apical periodontitis, a treatment plan involving guided EM with static navigation was implemented. Cone-beam computed tomography (CBCT) data was segmented to create a stereolithographic (STL) file of the tooth. This file was aligned and 3D-printed with a biocompatible resin to create a customized anatomical guide. The guide facilitated a precise osteotomy, accurate apex localization, and conservative flap management during surgery. The surgical procedure was completed successfully, with minimal complications. Follow-up CBCT one month later demonstrated excellent adaptation of the bone fragment and apical seal. Using 3D imaging and a customized anatomical guide in EM demonstrates promising outcomes for treating complex cases. The proposed design offers advantages by eliminating the need for guide tubes and facilitating conservative flap management. Further clinical studies are recommended to validate the long-term efficacy of this technique.