The European Food Safety Authority (EFSA) has updated its “Guidance on risk assessment of nanomaterials to be applied in the food and feed chain: human and animal health.” The updated guidance, published on August 3, 2021, covers the application areas within EFSA’s remit, including novel foods, food contact materials, food and feed additives, and pesticides. The guidance takes into account relevant scientific studies on physico‐chemical properties, exposure assessment and hazard characterization of nanomaterials, and areas of applicability. Together with the accompanying “Guidance on Technical requirements for regulated food and feed product applications to establish the presence of small particles including nanoparticles,” the updated guidance elaborates on physico‐chemical characterization, key parameters that should be measured, methods and techniques that can be used for characterization of nanomaterials, and their determination in complex matrices. The guidance also details aspects relating to exposure assessment and hazard identification and characterization. It discusses nanospecific considerations relating to in vitro/in vivo toxicological studies and outlines a tiered framework for toxicological testing. Furthermore, the guidance describes in vitro degradation, toxicokinetics, genotoxicity, and local and systemic toxicity, as well as general issues related to the testing of nanomaterials. According to the guidance, depending on the initial tier results, additional studies may be needed to investigate reproductive and developmental toxicity, chronic toxicity and carcinogenicity, immunotoxicity and allergenicity, neurotoxicity, effects on gut microbiome, and endocrine activity. The possible use of read‐across to fill data gaps, as well as the potential use of integrated testing strategies and the knowledge of modes or mechanisms of action, are also discussed. The guidance proposes approaches to risk characterization and uncertainty analysis.