Abstract Introduction Conventional sentinel node (SN) mapping is performed by injecting a radiocolloid followed by lymphoscintigraphy (and SPECT/CT imaging). An extra intraoperative injection with blue dye can then allow for optical identification of the SN. In order to improve the current clinical standard, the hybrid tracer indocyanine green (ICG)-99m Tc-nanocolloid was introduced, a tracer that is both radioactive and fluorescent. This feasibility study aimed to evaluate the value of a multimodal-based SN biopsy in vulvar cancer. Materials and methods Fifteen patients with vulvar cancer (29 groins) scheduled for SN biopsy were peritumorally injected with ICG-99m Tc-nanocolloid followed by lymphoscintigraphy and SPECT/CT imaging to identify the SNs. In thirteen patients, shortly before the start of the operation, blue dye was intradermally injected around the lesion. SNs were harvested using a combination of radiotracing, fluorescence imaging, and optical blue dye detection. A portable gamma camera was used before and after SN excision to confirm excision of the preoperatively defined SNs. Results Preoperative lymphoscintigraphy and SPECT/CT imaging visualized drainage to 39 SNs in 28 groins. During the operation, 98% ( ex vivo 100%) of the SNs were radioactive. With fluorescence imaging 96% of the SNs ( ex vivo 100%) could be visualized. Only 65% of the SNs had stained blue at the time of excision. Conclusion ICG-99m Tc-nanocolloid can be used for preoperative SN identification and enables multimodal (radioactive and fluorescent) surgical guidance in patients with vulvar cancer. The addition of fluorescence-based optical guidance offers more effective SN visualization compared to blue dye.