In oncology, sensitive and reliable detection tumor tissue is crucial to prevent recurrences and to improve surgical outcome. Currently, extensive research is focused on the use of radionuclides as well as fluorophores to provide real-time guidance during surgery to aid the surgeon in the identification of malignant tissue. Particularly, dual-modality approaches combining radionuclide and near-infrared fluorescence (NIRF) imaging have shown promising results in preclinical studies. Radionuclide imaging allows sensitive intra-operative localization of tumor lesions using a gamma probe, whereas NIRF imaging allows more accurate real-time tumor delineation. Consequently, both radionuclide and NIRF imaging might complement each other, and dual-modality image-guided surgery may overcome limitations of the currently used single-modality imaging techniques. In this review, a comprehensive overview on recent preclinical advances in tumor-targeted radionuclide and fluorescence dual-modality imaging is provided. Subsequently, the clinical applicability of dual-modality image-guided surgery is discussed.