Carbon-based nanomaterials have attracted much interest during the last decade for biomedical applications. Multimodal imaging probes based on carbon nano-onions (CNOs) have emerged as a platform for bioimaging because of their cell-penetration properties and minimal systemic toxicity. Here, we describe the covalent functionalization of CNOs with fluorescein and folic acid moieties for both imaging and targeting cancer cells. The modified CNOs display high brightness and photostability in aqueous solutions and their selective and rapid uptake in two different cancer cell lines without significant cytotoxicity was demonstrated. The localization of the functionalized CNOs in late-endosomes cell compartments was revealed by a correlative approach with confocal and transmission electron microscopy. Understanding the biological response of functionalized CNOs with the capability to target cancer cells and localize the nanoparticles in the cellular environment, will pave the way for the development of a new generation of imaging probes for future biomedical studies