Background: An ideal preoperative localization method is essential for the resection of small and deep-seated pulmonary nodules by video-assisted thoracoscopic surgery (VATS) in the era of low-dose computed tomography (CT) screening. This study describes a new localization method using electromagnetic navigation bronchoscopy (ENB) and compares it against conventional percutaneous CT-guided methods. Methods: Between January 2016 and May 2018, 18 consecutive patients with a total of 27 pulmonary nodules underwent ENB localization using patent blue vital dye before thoracoscopy for lung resection at the National Taiwan University Hospital. Over the same period, 268 patients had a total of 325 pulmonary nodules localized by a CT-guided method. Propensity analysis was applied to minimize bias during comparison. Results: Patients were selected using a propensity-score based process, matched for potential risk factors for localization failure, to ensure equal potential prognostic factors in both groups. After matching, the ENB group had 15 patients with a total of 24 pulmonary nodules, and the CT group had 30 patients with 48 pulmonary nodules. No major procedure-related complications occurred in either group. The target pulmonary nodule was not successfully localized for one patient in the ENB group and three in the CT group. The lesions were fully excised after conversion to mini-thoracotomy. Pathological examination confirmed the accuracy of the dye staining. Analysis found a non-significant difference in the success rate of these two localization methods. However, the following parameters were significantly different: interval between localization to surgery, global time, and rate of pneumothorax (p < 0.05). Conclusions: In the era of minimally invasive surgery, surgeons need an efficient one-step way to manage pulmonary nodules. Patent blue vital injection with ENB guidance in the operating room is a new, effective approach to localize small, deep-seated and non-palpable pulmonary lesions, comparable with CT-guided localization.