We analyzed the effects of levamisole on stress and the innate immune responses of pacu (Piaractus mesopotamicus). A total of 300 fish (180 ± 1.27 g) were fed a diet containing levamisole hydrochloride (LHC) for 15 days, then distributed into the following groups: T0 (control group); T1 (100), T2 (150), T3 (300) and T4 (500) mg kg−1 LHC (15 fish per group and four replicates per treatment). After this, fish (n = 8 per treatment) were exposed to air for three minutes to simulate stress conditions and were then challenged with the bacterium Aeromonas hydrophila to stimulate the immune system. Fish were sampled at 1, 3 and 24 h after bacterial inoculation to measure plasma cortisol and glucose concentrations, the leukocyte respiratory burst (LRB), hemolytic activity of the complement system (HAC50) and serum lysozyme activity (SLA). LHC attenuated the increase in plasma cortisol at 1 h (500 mg kg−1) and 3 h (300 mg kg−1) after air exposure and bacterial inoculation compared to control fish. The highest glucose concentrations were observed at 1 and 3 h after stress, which then returned to initial levels after 24 h, without any effect of LHC. The LHC 100 mg kg−1 dose increased LRB 1 h after inoculation and activated the HAC50 3 h later. At 24 h, all LHC concentrations increased the HAC50. SLA was reduced after inoculation, throughout the experimental period, without an effect of levamisole. Our results indicate that the oral administration of levamisole for 15 days modulated circulating cortisol levels during the stress response and improved the innate immune response against A. hydrophila infection in pacu.