Intraoperative radiography imaging is essential for accurate spinal implant placement. Hazards caused by ionizing radiation raised concern on personnel’s work life long exposure in the operating room (OR). To particularize a cumulative risk estimation of radiation of personnel and patient, depending on used methods (C-arm fluoroscopy, O-arm navigation) and patient characteristics during spinal surgery, detailed investigation of radiation exposure in a clinical setting is required. Lumbosacral dorsal spinal fusion was performed in 37 patients (19 navigated, 18 fluoroscopy) during this prospective study. Radiation exposure was measured on several body regions with thermoluminescent dosimeters on patient and OR personnel (surgeon, assistant, sterile nurse, radiology technologist). Comparison between patient characteristics and radiation exposure was included. The highest patients values were measured in the surgery field and gonads area during navigation (43.2 ± 19.4 mSv; fluoroscopy: 27.7 ± 31.3 mSv; p = 0.02), followed by the thoracic region during fluoroscopy (7.7 ± 14.8 mSv; navigation: 1.1 ± 1.0 mSv; p = 0.06), other measured regions can be considered marginal in comparison. Amongst OR personnel exposure of the surgeon was significant higher during fluoroscopy (right hand: 566 ± 560 µSv and thoracic region: 275 ± 147 µSv; followed by thyroid and forehead) compared to navigation (right finger: 49 ± 19 µSv; similar levels for all regions; p < 0.001 in all regions). When compared to the surgeon, other OR personnel had significantly lower radiation doses on all body regions using fluoroscopy, and similar dose during navigation. The highest eye's lens region value was measured during fluoroscopy for the patient (185 ± 165 µSv; navigation: 205 ± 60 µSv; p = 0.57) and the surgeon (164 ± 74 µSv; navigation: 92 ± 41 µSv; p < 0.001). There was a significant correlation between patient BMI and radiation exposure to the surgery field during fluoroscopy. To our knowledge, these data present the first real life, detailed comparison of radiation exposure on OR personnel and patients between clinical use of navigation and fluoroscopy. Although patient's radiation dose is approximately 3-fold during navigation compared to the fluoroscopy, we found that a spinal surgeon could perform up to 10-fold number of surgeries (10.000 versus 883) until maximum permissible annual effective radiation dose would be reached. Especially for a spinal surgeon, who is mainly exposed amongst OR personnel, radiation prevention and protection must remain a main issue.