Navigation bronchoscopy is an emerging technique used to evaluate pulmonary lesions. Using Veran’s SPiN electromagnetic navigation bronchoscopy (ENB) and Archimedes virtual bronchoscopy navigation (VBN), this study aimed to compare the accuracy and safety of these procedures for lung lesions and to identify potentially relevant knowledge for the application of artificial intelligence in interventional pulmonology in a single institute. This single-center, retrospective study compared the ENB and VBN results in patients with pulmonary lesions unsuitable for biopsy via percutaneous transthoracic needle biopsy methods. A total of 35 patients who underwent navigation bronchoscopy for pulmonary lesion diagnosis were enrolled. Nineteen patients were stratified in the ENB group, and sixteen were in the VBN group. The mean age of this cohort was 67.6 ± 9.9 years. The mean distance of the lesion from the pleural surface was 16.1 ± 11.7 mm (range: 1.0–41.0 mm), and most lesions were a solid pattern (n = 33, 94.4%). There were 32 cases (91.4%) of pulmonary lesions with an air-bronchus sign. A statistically significant difference was found between pulmonary size and transparenchymal nodule access (p = 0.049 and 0.037, respectively). The navigation success rate was significantly higher in the VBN group (93.8% vs. 78.9%). Moreover, no procedure-related complications or mortality were noted. The radiographic characteristics, such as size or solid component, can affect the selection of the biopsy procedure, either ENB or VBN. Navigation bronchoscopy-guided biopsy demonstrated acceptable accuracy and a good safety profile in evaluating pulmonary lesions when the percutaneous approach was challenging or life threatening.