The demand for food has intensified production in agricultural areas and stimulated the use of nanotechnology to develop new inputs, especially nanoparticle materials. In this new context, predicting the impact of using nanoparticles on non‐target organisms becomes a necessary measure. The aim of this study was to evaluate the ecotoxicological potential of magnesium (Mg2+) added via magnesium oxide nanoparticles (MgO‐NPs), magnesium oxide (MgO), and magnesium nitrate hexahydrate (Mg [NO3]2·6H2O) incubated over time in tropical soil on earthworms (Eisenia andrei), springtails (Folsomia candida), and enchytraeids (Enchytraeus crypticus). Tests were conducted using a clay‐textured Latossolo Vermelho distrófico (Oxisol), which received increasing doses of Mg2+ (0; 25; 50; 100; 200 and 400 mg kg−1 of soil) from the three sources tested added to the soil. Treated soil was incubated for 120 days in a room with controlled temperature and photoperiod, and the ecotoxicological tests were performed at 0, 60, and 120 days of incubation. Despite having caused reduction in the reproduction of F. candida at the incubation time 0, MgO‐NPs showed a low toxic potential against the other species studied, with toxicity only at a higher dose of 50 mg Mg kg−1 when compared to the other sources of Mg2+ applied to the soil (MgO and Mg [NO3]2·6H2O). Responses associated with incubation times showed that all magnesium sources tested have lower toxicity over incubation time.