The aim of the study was to assess the quality, accuracy and benefit of navigated 2D and 3D ultrasound for intra-axial tumor surgery in a prospective study. Patients intended for gross total resection were consecutively enrolled. Intraoperatively, a 2D and 3D iUS-based resection was performed. During surgery, the image quality, clinical benefit and navigation accuracy were recorded based on a standardized protocol using Likert’s scales. A total of 16 consecutive patients were included. Mean ratings of image quality in 2D iUS were significantly higher than in 3D iUS (p < 0.001). There was no relevant decrease in rating during the surgery in 2D and 3D iUS (p > 0.46). The benefit was rated 2.2 in 2D iUS and 2.6 in 3D iUS (p = 0.08). The benefit remained stable in 2D, while there was a slight decrease in the benefit in 3D after complete tumor resection (p = 0.09). The accuracy was similar in both (mean 2.2 p = 0.88). Seven patients had a small tumor remnant in intraoperative MRT (mean 0.98 cm3) that was not appreciated with iUS. Crucially, 3D iUS allows for an accurate intraoperative update of imaging with slightly lower image quality than 2D iUS. Our preliminary data suggest that the benefit and accuracy of 2D and 3D iUS navigation do not undergo significant variations during tumor resection.