Graphene-like (GL) layers, a new graphene-related material (GRM), possess peculiar chemical, colloidal, optical and transport properties. Considering the very recent promising application of GL layers in biomedical and bioelectronic fields, it is of utmost importance to investigate the toxicological profile of these nanomaterials. This study represents an important first report of a complete in vivo toxicity assessment of GL layers on embryonic zebrafish (Danio rerio). Our results show that GL layers do not lead to any perturbations in the different biological parameters evaluated, indicating their good biocompatibility on a vertebrate model. The new insight into the biosafety of GL layers will expand their applications in nanomedicine.