Intraoperative visualization of molecular processes delineated by fluorescence contrast agents offers the potential for increased surgical precision and better patient outcomes. To exploit fully the clinical potential for targeted fluorescence guidance, there is a growing need to develop high-resolution, quantitative imaging systems suitable for surgical use. Diffuse optical fluorescence tomography (DOFT) systems in pre-clinical and diagnostic imaging applications have demonstrated improvements in fluorescence quantification with the addition of a priori data from structural imaging modalities (e.g., MR, CT). Here, we investigate the use of a cone-beam CT (CBCT) surgical guidance system to generate spatial priors for intraoperative DOFT. Imaging and localization data is incorporated directly into a finite element method DOFT implementation (NIRFAST) at multiple stages. First, CBCT data from an intraoperative flat-panel C-arm is used to generate tetrahedral meshes. Second, optical tracking of laser and camera devices enables an adaptable non-contact DOFT approach to accommodate various anatomical sites and acquisition geometries. Finally, anatomical segmentations from CBCT are included in the optical reconstruction process using Laplacian-type regularization (“soft spatial priors”). Calibration results showed that light rays between the tissue surface and navigated optical devices were mapped with sub-millimeter accuracy. Liquid phantom experiments determined the improvements in quantification of fluorescence yield, with errors of 85% and <20% for no priors and spatial priors, respectively. CBCT-DOFT fusion in a VX2-tumor rabbit model delineated contrast enhancement using a dual CT/optical liposomal nanoparticle. These developments motivate future translation and evaluation in an ongoing CBCT-guided head and neck surgery patient study.