Abstract Background The study aims to assess the accuracy of multi-parametric prostate MRI (mpMRI) and 18F-choline PET/CT in tumor segmentation for clinically significant prostate cancer. 18F-choline PET/CT and 3 T mpMRI were performed in 10 prospective subjects prior to prostatectomy. All subjects had a single biopsy-confirmed focus of Gleason ≥ 3+4 cancer. Two radiologists (readers 1 and 2) determined tumor boundaries based on in vivo mpMRI sequences, with clinical and pathologic data available. 18F-choline PET data were co-registered to T2-weighted 3D sequences and a semi-automatic segmentation routine was used to define tumor volumes. Registration of whole-mount surgical pathology to in vivo imaging was conducted utilizing two ex vivo prostate specimen MRIs, followed by gross sectioning of the specimens within a custom-made 3D-printed plastic mold. Overlap and similarity coefficients of manual segmentations (seg1, seg2) and 18F-choline-based segmented lesions (seg3) were compared to the pathologic reference standard. Results All segmentation methods greatly underestimated the true tumor volumes. Human readers (seg1, seg2) and the PET-based segmentation (seg3) underestimated an average of 79, 80, and 58% of the tumor volumes, respectively. Combining segmentation volumes (union of seg1, seg2, seg3 = seg4) decreased the mean underestimated tumor volume to 42% of the true tumor volume. When using the combined segmentation with 5 mm contour expansion, the mean underestimated tumor volume was significantly reduced to 0.03 ± 0.05 mL (2.04 ± 2.84%). Substantial safety margins up to 11–15 mm were needed to include all tumors when the initial segmentation boundaries were drawn by human readers or the semi-automated 18F-choline segmentation tool. Combining MR-based human segmentations with the metabolic information based on 18F-choline PET reduced the necessary safety margin to a maximum of 9 mm to cover all tumors entirely. Conclusions To improve the outcome of focal therapies for significant prostate cancer, it is imperative to recognize the full extent of the underestimation of tumor volumes by mpMRI. Combining metabolic information from 18F-choline with MRI-based segmentation can improve tumor coverage. However, this approach requires confirmation in further clinical studies.