Nano-world is expanding rapidly due to the amalgamation of different science disciplines. Nanomaterials (NMs) have wide applications in the biomedical field due to their unique properties like the ability to investigate substances at the molecular level, growth-enhancing ability, drug carriers, fillers, and catalysts. However, NMs are extensively used in the biomedical field would possibly lead to several complications in human health that limit their present applications. This scenario highlighted an urgent need to investigate, examine and find out the proper solutions that can minimize the risk associated with the manufacture and use of NMs in the biomedical field. A large number of complicated interactions between NMs and biological systems show that there is no universal toxicity mechanism or assessment method for NMs. The main purpose of this review is to focus on the toxicity of NMs; their safety aspects and different molecular mechanisms of nanotoxicity. The role of various factors that influences nanotoxicity like size, morphology, surface charges, other surface properties, solubility, pH, light activation, and modes of interactions has been explained briefly. Moreover, ideal properties of NMs required for biomedical applications like biocompatibility, antigenicity, clearance rate, surface properties, morphology, mode of drug delivery, drug solubility, and bioavailability have been discussed in detail. The invitro, in-vivo, and in-silico assessments for nanotoxicity are narrated briefly in this review along with the cytotoxicity mechanisms of NMs. A special focus is given to the recent development in toxicological assessment of NMs, and safety guidelines. These developments and safety guidelines play an important role to minimize toxicity and promote the use of the NMs in the biomedical field.