Dye-doped near infrared-emitting silica nanoparticles (DD-NIRsiNPs) represent a valuable tool in bioimaging, because they provide sufficient brightness, resistance to photobleaching and consist of hydrophilic non-toxic materials. Here, we report the development of multiple dye-doped NIR emitting siNPs (mDD-NIRsiNPs), based on silica–PEG core–shell nanostructures doped with a donor–acceptor couple, exhibiting a tunable intensity profile across the NIR spectrum and suitable for both multiparametric flow cytometry analyses and time-domain optical imaging. In order to characterize the optical properties and fluorescence applications of the mDD-NIRsiNPs, we have characterized their performance by analyzing their in vivo biodistribution in healthy mice as well as in lymphoma bearing xenografts, and their suitability as contrast imaging agents for cell labeling and tracking. The mDD-NIRsiNPs features will be useful in designing new applications for imaging agents based on silica nanoparticles for different experimental disease models.