The aim of this study was to evaluate simultaneously a battery of biomarkers in Prochilodus lineatus fish in order to obtain a holistic overview of silver nanoparticles (AgNP) toxicity. Juveniles were exposed to 2.5 and 25.0 µgAgNP L−1 for 5 and 15 days. We analyzed silver accumulation in tissues (liver, intestine and brain), morphometric and hematological parameters, oxidative stress markers (antioxidant enzymes, lipid peroxidation, and antioxidant capacity against peroxyl radicals-ACAP-), and metabolic responses in plasma (glucose, proteins, triglycerides and cholesterol), liver and muscle (glycogen, proteins, and lipids). Additionally, after 15-day exposure, the number of colony forming units (CFU) of bacteria living at the skin mucus secretion was analyzed to corroborate the AgNP biocide properties. Tissue Ag accumulation was concentration- and time-dependent: liver > intestine > brain. The hepatosomatic index increased at both periods of exposure at 25.0 µgAgNP L−1. Hematological and plasma biochemical parameters increased after 5 days of exposure to the highest concentration. When fish were exposed for 15 days to 25.0 µgAgNP L−1, many changes were observed: glycogen content increased in liver and muscle, muscle protein concentration decreased, all antioxidant enzymes activities were enhanced and ACAP decreased in liver. The CFU decreased in the mucus of fish exposed to both AgNP treatments when compared to the control group. The multivariate analysis revealed a clear difference in physiological profiles of individuals exposed to the different treatments. Our results showed that AgNP in short and subchronic exposures threaten the health of fish.