The objective of this work was to evaluate the ecotoxicological qualitative risk associated with the use of sewage sludge containing Zn oxide nanoparticles (ZnO-NPs) as soil amendment. A sludge-untreated soil and two sludge-treated soils were spiked with ZnO-NPs (0-1,000 mg/kg soil). Soil ecotoxicity was assessed with Eisenia fetida (acute and sublethal end points), and the unfilterable and filterable (0.02 μm) soil leachates were tested with a battery of biomarkers using Chlorella vulgaris, Daphnia magna, and the fish cell line RTG-2 (Oncorhynchus mykiss). The production of E. fetida cocoons in sludge-treated soils was lower than that in sludge-untreated soils. The highest effect in the algal growth inhibition test was detected in sludge-untreated soil, most likely caused by the loss of organic matter in these samples. The D. magna results were always negative. Toxic effects (lysosomal cell function and production of reactive oxygen species) in RTG-2 cells were only observed in sludge-treated soils. In general, the toxicity of ZnO-NPs in sludge-treated soils was similar to that of sludge-untreated soil, and the filterable leachate fraction [Zn salt (Zn(2+))] did not produce greater effects than the unfilterable fraction (ZnO-NPs). Thus, after the addition of ZnO-NP--enriched sewage sludge to agricultural soil, the risk of toxic effects for soil and aquatic organisms was shown to be low. These findings are important because repeated use of organic amendments such as sewage sludge may cause more and more increased concentrations of ZnO-NPs in soils over the long-term.