Carbon nano-onions (CNOs), together with graphene and its derivatives, are one of the most interesting carbon nanostructures due to their peculiar chemical and physical properties. Made of a number of concentric fullerene layers, carbon-based structures have a peculiar design and assume the appearance of onion-like cages. Due to their excellent biocompatibility and safety, CNOs have low toxicity, high water dispersibility (due to surface functionalization), and high pharmacological efficacy. These properties render them highly appealing for applications such as drug delivery, sensing, imaging, tissue engineering, and therapeutic agents. While CNOs were discovered almost at the same time as other carbon nanomaterials (CNMs), their potential in biological applications remains largely unexplored. On the other hand, similar to other CNMs and fullerenes, CNOs play a crucial role as they represent carbon's ability to form diverse nanostructures with exceptional properties. This review aims to summarize recent studies on CNOs for biological applications, underlining the current achievements, possible opportunities, and challenges toward future development.