After more than a decade of studying the ecotoxicity of graphene oxide nanomaterials (nGOs), it has been concluded that there is limited information available regarding the environmental risk of graphene-based materials. Since existing ecotoxicological studies of nanomaterials have produced contradictory results, it is recommended that case-by-case studies should be conducted to evaluate their effects. This can be carried out by employing several methods, testing species from different trophic levels, and conducting community studies. Our goal was to evaluate the toxicity effects of two GOs (AF 96/97 and PM 995) derived from different graphite precursors on various test organisms from diverse trophic levels (bacteria, protozoa, a freshwater microbial community, plants, and invertebrate animals) in aquatic environments. We compared the effects of both nGO types and estimated the predicted no-effect environmental concentration (PNEC) values to determine their potential environmental risk. Our findings demonstrated the need for a complex ecotoxicity toolkit since the ecotoxicity results varied based on the test organism, the selected endpoints, and the test method used. Additionally, we found that toxicity effects were dependent on the concentration and characteristics of the specific nGO type used, as well as the exposure time. We estimated the PNEC values for GO AF 96/97 and GO PM 995 in the aquatic compartment to be 8 ng/L and 4 ng/L, respectively. Even after applying the worst-case scenario approach, the tested nGOs pose no environmental risk.