Graphene oxide (GO), owing to its extraordinary application prospects in biomedicine, is attracting growing research attention. However, the biosafety and blood compatibility of GO required for its clearance for use in clinical trials remain elusive. Therefore, we studied the mutagenic properties of GO as well as its cell toxicity and blood compatibility. Prior to biological experiments, we assessed the structural organization of GO using dynamic light scattering and microscopic visualization methods. The results of both the Ames mutagenicity test performed on Salmonella enterica serovar Typhimurium TA98 and TA102 strains and the cytotoxicity test on noncancerous, immortalized human keratinocytes revealed no mutagenic or toxic effects of GO. Simultaneously, GO reduced the viability of the MelJuSo human melanoma cell line. A blood compatibility assay revealed that a concentration of 10 μg/mL was critical for GO biosafety, as greater concentrations induced diverse side effects. Specifically, GO disrupts erythrocytes’ membranes in the dose-dependent manner. Moreover, GO at higher concentrations both inhibited the process of ADP (a physiological platelet agonist)-induced cell aggregation and affected their disaggregation process in platelet-rich plasma. However, in the blood clotting assessment, GO showed no effects on the activated partial thromboplastin time, prothrombin time, or thrombin time of the platelet-poor plasma. The obtained results clearly indicate that the relationship between the GO preparation method, its size, and concentration and biosafety must be cautiously monitored in the context of further possible biomedical applications.