Intraoperative guidance using targeted near-infrared (NIR) fluorescent tracers can provide surgeons with real-time feedback on the presence of residual tumour tissue. To overcome the still limited depth penetration of NIR light, and limit potentially missing residual occult or deeper lying lesions, the combination of fluorescence with nuclear imaging is proposed. We describe the design and preclinical validation of the anti-HER2 nanobody 2Rs15d, conjugated with a ‘multifunctional single attachment point’ (MSAP), which integrates a Cy5 fluorophore and diethylenetriaminepentaacetic acid (DTPA) chelator into a single label. After random conjugation to primary amines in the nanobody, functionality of the tracer and stability after 111In labelling were evaluated in vitro. Using SKOV3 (HER2+) and MDA-MB-435S (HER2-) xenografted mice, the in vivo biodistribution of 2Rs15d-MSAP.111In was determined by SPECT/CT (1h post-injection) and fluorescence imaging (1h30 post-injection). Ensuing, the ex vivo biodistribution was determined 2h (both xenograft models) and 24h post-injection (SKOV3 only). The tracer retained its affinity after conjugation of the MSAP and remained stable over 24h in both PBS and human serum after 111In labelling. The in vivo SPECT/CT and fluorescence images corresponded well, showing the expected biodistribution pattern for nanobody tracers, meaning low background except for high renal uptake due to clearance, and specific tumour uptake in HER2-overexpressing tumours. Ex vivo biodistribution data revealed a SKOV3 tumour-specific uptake of 7.0 ± 2.5 %ID/g after 2h, significantly higher than 1.1 ± 1.2 %ID/g for control tumours. The tumour-to-blood ratio was 47.6± 25.4, tumour-to-muscle ratio 23.2 ± 11.6, and tumour-to-liver ratio 6.9 ± 3.7. After 24h SKOV3 tumour uptake was 5.6 ± 1.9 %ID/g, tumour-to-blood ratio 229.1 ± 85.1, tumour-to-muscle ratio 16.8 ± 8.0, and tumour-to-liver ratio 5.1 ± 1.9. In conclusion, functional bimodal nuclear/fluorescent nanobody-tracers can be conveniently generated by conjugation of a single-molecule MSAP-reagent carrying both fluorophore and a chelator.