Nanomaterials have been used in a wide range of biomedical research fields, such as delivery of biomolecules, biosensing, bioimaging, and tissue scaffolding. These materials have also emerged in some biotechnology products and in preclinical/clinical trials for humans. Increasing use of nanomaterials in science and technology and consequently their wide exposure to the environment and public health motivate the development of reliable toxicity assays and accurate physicochemical characterization of nanomaterials. This work outlines commonly used methodologies to evaluate the nanomaterial toxicity in vitro and in vivo. Some studies were then discussed regarding the toxicity of various nanomaterials (i.e., metallic nanoparticles, metal oxides, carbon-based nanomaterials, and quantum dots) exposed to cells, tissues, animal models, and humans. A diverse nature of nanotoxicity assays and nanomaterials has raised some unpredictable and somehow contradictory nanotoxicity results about the safe use of existing or newly developed nanomaterials.