The introduction of optical imaging by using near-infrared (NIR) light shines new light in the field of (oncologic) surgery. The use of non-specific fluorophores, such as Indocyanine Green (ICG) and Methylene Blue (MB) have already shown its value for different applications during image-guided surgery. Both ICG and MB are currently the only fluorophores approved by regulatory agencies for off-label use. ICG improves visibility of several solid tumors, sentinel lymph nodes, biliary ducts and can be used to evaluate tissue perfusion. MB could be used for ureter imaging and neuroendocrine or thyroid tumors detection. Recently, a shift to molecular imaging was made by the introduction of new NIR fluorophores (IRDye-800CW, ZW800-1), which could be conjugated to tumor or structure specific targets, such as proteins, antibodies, antibody fragments or nanoparticles. Several clinical trials showed detection of both tumor and metastases in patients with head-and neck, colorectal, pancreatic, ovarian, and renal tumors. Furthermore, nerve and ureter specific agents are (pre-)clinically evaluated, however more research is necessary to make these agents clinically available. Limitations of using NIR fluorescence imaging during surgery are the lack of quantification of fluorescence signals and limited penetration depth. Further optimization of NIR fluorescence imaging and evaluation of the clinical benefit for the patient are necessary steps to make NIR fluorescence guided surgery general applicable into surgical daily practice.