Incorporating iron nanoparticles into graphene oxide (GO) may enhance its potential for use in various applications. However, alterations to the GO structure could pose a risk to environmental organisms and should therefore be fully understood before their further use. In this paper, we prepared iron-doped graphene oxide from pure graphene oxide and two different iron sources with iron in two different oxidation states. Prepared samples were characterized in detail by SEM, EDS, XRF, Raman spectroscopy, XPS, and TEM. In the next step, these samples were subjected to ecotoxicological evaluation in three model organisms: mustard Sinapis alba, freshwater algae Desmodesmus subspicatus, and saltwater crustaceans Artemia salina. Our results showed a stimulatory effect of iron-doped GO on S. alba seeds and a modest degree of growth inhibition for D. subspicatus when compared to pure GO at a concentration of 100 mg/L. In the case of A. salina, mortality was observed at a concentration of 10 mg/L for all tested nanoparticles. However, the iron-doped nanoparticles exhibited a more than twofold decrease in mortality. Our findings suggest that iron-doped GO have a reduced toxicity compared to pure GO, but further research is necessary to enhance the understanding of their behaviour in the environment.