Even though nanoparticles (NPs) are mostly removed by wastewater treatment plants, wastewater-borne NPs may show an altered toxicity to aquatic organisms. The main objectives of this work were: i) to assess the chronic (28 days) effects of wastewater-borne NPs of silver (AgNPs, 1.4-36.2 μg L-1) and titanium dioxide (TiO2NPs, 3.1-50.2 μg L-1) at the individual (growth) and biochemical (biomarkers of neurotoxicity, oxidative stress and energy metabolism) levels in rainbow trout Oncorhynchus mykiss; and ii) to compare them with their effluent-supplemented and water-dispersed counterparts. The total Ag and Ti levels were determined in several fish organs. The growth of O. mykiss was not affected by the NPs in any treatment, except a 29% increase at 5.5 μg L-1 of total Ag supplemented to effluents. The Ag level in organs of O. mykiss was significantly higher after exposure to water-dispersed AgNPs than their wastewater-borne or effluent-supplemented counterparts. No significant Ti uptake could be observed. Effluent-supplemented TiO2NPs (50.1 μg L-1 Ti) potentially induced neurotoxic effects, indicated by a 24% increase in acetylcholinesterase activity comparatively to controls. Energy reserves were unaffected by TiO2 treatments, while nearly all AgNP-containing treatments caused a depletion of total lipids, proteins and carbohydrates in the muscle, suggesting an increased energy demand for detoxification processes to cope with AgNPs. Besides NPs, the effluent matrix and dispersing agent (for AgNPs) induced significant effects on energetic reserves and oxidative stress, indicating background toxicity of both treatments at the biochemical level. Our study is the first to assess chronic effects of wastewater-borne NPs on rainbow trout. While no effects were found at the individual level, several biochemical markers were changed by the NPs exposure. Our results highlight the importance of using complex matrices for a reliable risk assessment of NPs in the aquatic environment.