The aim of this study was to clinically assess the impact of a dental operating microscope (DOM) on the clinician's ability to locate a second canal in the mesiobuccal root (MB2), also known as the mesiolingual canal. An endodontic specialist treated 180 maxillary first molars using the same protocol for all teeth except for the use of the DOM (n = 90 per group). In the teeth treated without the use of a DOM, a mesiolingual canal was located in 26.67% of the cases; when a DOM was used, a mesiolingual canal was located in 77.78% of the cases. Analysis with the Fisher exact test revealed that the magnification provided by a DOM significantly (P < 0.05) improved the identification of MB2 canals in maxillary first molars.