Objectives This study aims to evaluate the feasibility of a custom robot system guided by optical cone beam computed tomography (CBCT)-based navigation for skull base biopsy. Design An accuracy study was conducted. Setting Platform for navigation and robot-aided surgery technology. Participants Phantom skull. Main Outcome Measures The primary outcome measure was to investigate the accuracy of robot-assisted needle biopsy for skull base tumors. A 14-gauge needle was automatically inserted by the five degrees of freedom robot into the intended target, guided by optical navigation. The result was displayed on the graphical user interface after matrix transformation. Postoperative image scanning was performed, and the result was verified with image fusion. Results All 20 interventions were successfully performed. The mean deviation of the needle tip was 0.56 ± 0.22 mm (measured by the navigation system) versus 1.73 ± 0.60 mm (measured by image fusion) ( p < 0.05). The mean insertion depth was 52.3 mm (range: 49.7-55.2 mm). The mean angular deviations off the x-axis, y-axis, and z-axis were 1.51 ± 0.67, 2.33 ± 1.65, and 1.47 ± 1.16 degrees, respectively. Conclusions The experimental results show the robot system is efficient, reliable, and safe. The navigation accuracy is a significant factor in robotic procedures.