Seminal cryopreservation allows the long-term conservation of gametes of various species, including endangered species, such as Prochilodus brevis. However, the application of this biotechnology can cause damage to sperm cells, reducing seminal quality. Thus, we have sought substances that minimize the damage caused by this process, such as antioxidants. Thus, this study aimed to evaluate the association between two cryoprotectants and two vitamins, in different concentrations, on the quality of cryopreserved semen of P. brevis. For cryopreservation, the experiment was performed in two stages. In the first stage, the semen of 10 animals was submitted to six different freezing means, coming from the combination of 5% glucose, two cryoprotectants (Dimethyl sulfoxide [DMSO] or Methyl glycol) and two vitamins (C or E to 0.0001 mg) for cryopreservation. In the second stage, semen samples of eight animals were diluted in 5% glucose and the best cryoprotectant found in the first stage, associated with three different concentrations of vitamins C or E (0.01, 0.001, and 0.0001 mg). In both steps, the in natura and post-thawed samples were submitted to kinetic analysis, morphology, and sperm membrane integrity. The cryopreserved semen with DMSO presented significantly higher results (p < 0.05) than that frozen with Methyl glycol, regardless of the vitamin used. The morphologically normal spermatozoa rate was higher (p < 0.05) in the vitamin-containing samples, however, vitamin E reduced sperm motility rates, independent of the cryoprotectant used. As for vitamin concentrations, higher motility rates were obtained when cryopreserved semen with 0.01 and 0.0001 mg of any of the vitamins. However, the higher concentration had a deleterious effect on the spermatic morphology of P. brevis. Therefore, the glucose associated with DMSO and the lower concentration of vitamin C provides good quality for the post-thawed semen of P. brevis.