Intraoperative definition of tumor free resection margins in head and neck cancer is challenging. In the current proof-of-principle study we evaluated a novel silica nanoparticle-based agent for its potential use as contrast enhancer. We synthesized silica nanoparticles with an average size of 45 nm and modified these particles with the fluorescence stain fluorescein isocyanate (FITC) for particle detection and with epidermal growth factor receptor (EGFR)-targeting antibodies for enhanced tumor specificity. The nanoparticles exhibited good biocompatibility and could be detected in vitro and in vivo by confocal laser scanning microscopy. Additionally, we show in an ex vivo setting that these modified nanoparticles specifically bind to tumor samples and could be detected using a handheld confocal fluorescence endomicroscope. From a clinical point of view, we believe that this method could be used for tumor border contrast enhancement and for better intraoperative definition of R-0 tumor resection.