Pacu stands out due to its favorable characteristics for captive rearing, such as hardiness, rapid growth, high consumer market acceptance, and especially its omnivorous feeding habit, which supports its cultivation in biofloc systems (BFT). The BFT system offers high water efficiency, biosafety, and significantly reduces the environmental impact caused by aquaculture activities. This system is formed by stimulating microorganisms in the water through manipulation of the carbon/nitrogen (C/N) ratio. As a result, part of the nitrogenous compounds in the culture water—originating from feed waste and feces—is recycled. However, BFT systems tend to accumulate nitrate (NO₃⁻) over production cycles, thereby altering the culture environment. Nitrate is toxic to farmed organisms and, in chronic exposure, can cause sublethal effects on aquatic organisms. Therefore, the objective of this study was to evaluate the effects of different nitrate levels on zootechnical performance, blood parameters, and oxidative stress in juvenile pacu reared in a BFT system, as well as to assess the bacterial composition of the biofloc. The experiment was conducted at the Continental Aquaculture Laboratory (LAC) of the Federal University of Rio Grande (FURG) over a 45-day period. The biofloc was established using a concentrated inoculum and adjusted to achieve a total suspended solids (TSS) concentration of 250 mg L⁻¹, maintaining a C/N ratio of 15:1. Treatments were designated as BFT150, BFT300, and BFT500, with nitrate concentrations of 139.51 ± 4.28; 333.66 ± 11.94; and 519.11 ± 18.83 mg NO₃-N L⁻¹, respectively. Fish were randomly distributed (9 fish per tank; total = 108 juveniles, 31.34 ± 0.14 g) in 310 L tanks (250 L effective volume) with continuous aeration and controlled temperature (27°C). Fish were fed twice daily (9 a.m. and 4 p.m.) at 3% of biomass with a commercial diet containing 36% crude protein. During the experiment, samples of biofloc, blood, and tissue (9 fish per treatment) were collected. Water quality parameters remained within optimal ranges for the species and system throughout the study. After 45 days, it was observed that only BFT500 negatively affected zootechnical performance. Hematocrit and hemoglobin levels showed significant differences, with BFT150 showing the greatest alterations; for erythrocytes, only BFT500 differed significantly, presenting the lowest values. In terms of oxidative stress biochemical parameters, gill tissue exhibited high protein and lipid peroxidation (LPO) at BFT150. Glutathione-S-Transferase (GST) activity differed significantly across all treatments (BFT300 > BFT500 > BFT150), and lactate levels were higher in BFT300 and BFT500 compared to BFT150. In liver tissue, antioxidant capacity against peroxyl radicals (ACAP) was higher in BFT500, while thiol protein content (P-SH) was lower in BFT300. Glucose levels were reduced in BFT500, and lactate was lower in BFT300. The bacterial community showed no significant differences except for free filaments and Vibrio. An increase in coccoid populations, aggregated filamentous bacteria, and bacilli was observed, along with a reduction in free filamentous bacteria and Vibrio over time. Based on these findings, it is concluded that juvenile pacu should be cultured at nitrate concentrations below BFT300, as even this level caused alterations in several evaluated parameters, which should be avoided to ensure optimal growth and development.