Background and Objectives: As brain lesions present complex diagnostic challenges, accurate tissue sampling via biopsy is critical for effective treatment planning. Traditional frame-based stereotactic biopsy has been complemented by navigated biopsy techniques, leveraging advancements in imaging and navigation technology. This study aims to compare the navigated and frame-based stereotactic biopsy methods in a clinical setting, evaluating their efficacy, safety, and diagnostic outcomes to determine the optimal approach for precise brain lesion targeting. Materials and Methods: retrospective analysis was conducted on patients who underwent brain biopsies between January 2017 and August 2023 at an academic medical center. Data on patient demographics, clinical characteristics, biopsy technique (navigated vs. frame-based), and outcomes including accuracy, complications, and hospital stay duration were analyzed. Results: The cohort comprised 112 patients, with no significant age or gender differences between groups. Symptoms leading to biopsy were predominantly diminished muscle strength (42.0%), cognitive issues (28.6%), and aphasia (24.1%). Tumors were most common in the deep hemisphere (24.1%). The median hospital stay was 5 days, with a rehospitalization rate of 27.7%. Complications occurred in 4.47% of patients, showing no significant difference between biopsy methods. However, navigated biopsies resulted in fewer samples (p < 0.001) but with comparable diagnostic accuracy as frame-based biopsies. Conclusions: Navigated and frame-based stereotactic biopsies are both effective and safe, with comparable accuracy and complication rates. The choice of technique should consider lesion specifics, surgeon preference, and technological availability. The findings highlight the importance of advanced neurosurgical techniques in enhancing patient care and outcomes.