Nanotechnology has emerged as a key discipline in biomedical innovation, owing to the unique properties that materials exhibit at the nanoscale. These characteristics have enabled its application in the diagnosis, prevention, and treatment of diseases in both humans and animals. In human health, nanomaterials have significantly enhanced the precision of imaging diagnostic techniques, facilitated the regeneration of oral and skin tissues, and optimized the targeted delivery of drugs and bioactive compounds. In veterinary medicine, notable advances include improved animal reproduction through hormonal delivery systems and sperm purification, enhanced nutrition via nanoencapsulation of nutrients and phytocompounds, and the development of high-sensitivity diagnostic biosensors. The physicochemical characterization of nanomaterials including size, shape, and surface charge is essential for understanding their biological behavior. From an integrative perspective, nanotechnology offers strategic opportunities to address interconnected challenges across human, animal, and environmental health, in alignment with the One Health approach of the 2030 Agenda. Its responsible application can contribute to food security, zoonosis control, and the sustainable use of resources, thereby promoting resilient and sustainable health systems. Nevertheless, its implementation faces major challenges, including toxicity, biocompatibility, regulatory compliance, and industrial scalability. This chapter provides a critical review of the principles, current applications, and limitations of nanotechnology in human and animal health, aiming to support the development of innovative, safe, and effective strategies that embrace a transdisciplinary vision of global health.