A preservation protocol has not been established for Colossoma macropomum oocytes, and its development may improve the production and breeding programs of this South American fish species. Thus, this study aimed to determine the effect of different methods and protocols for the preservation of C. macropomum oocytes. Seven experiments were conducted throughout the breeding season of this species. The oocytes were collected and stored in sterile conditions. Preserved oocytes were subjected to storage in the following treatments: room temperature (RT, 27 °C), centrifugation followed by ovarian fluid removal (Cen), vacuum (Vac), chilled temperature (ChT), centrifugation and vacuum (CV), vacuum and chilled temperature (VChT), and centrifugation, vacuum, and chilled temperature (CVChT) in dry sterilized plastic containers and plastic bags. Chilled storage was tested at 4 and 13 °C. Fertilization and hatching rates were assessed at 0, 30, 60, 90, and 120 min after stripping (MAS) for preservation protocols. The larval malformation rate was analyzed at 0 and 30 MAS for RT and ChT. Quantitation and identification (by mean of MALDI-TOF MS) of bacteria were performed at 0, 60, 90, and 120 MAS, and scanning electron microscopy (SEM) analyses were carried out at 0, 60, and 90 MAS. The fertilization and hatching rates decreased over preservation time and breeding season. RT samples fertilized at 0, 30, and 60 MAS yielded similar fertilization rates at both the beginning and end of the season. By the end of the season, oocytes from treatment ChT at 13 °C 30 MAS yielded higher fertilization and hatching rates, and a lower percentage of larvae malformation than RT 30 MAS. The treatment ChT at 4 °C triggered low a fertilization rate. The treatments ChT (13 °C) and Cen provided good fertilization rate when used alone and with other approaches, i.e., treatments VChT, CV, and CVChT. The treatment Vac presented inconsistent results, so no conclusion could be made. Bacterial colony counts were low (10-1.6 × 10 CFU-mL), and a total of 18 bacteria species were identified in all batches analyzed; however, the treatments did not influence the number of bacteria. C. macropomum female breeders presented distinct bacteria species in their oocytes and the presence of bacteria did not impair oocyte quality until 120 MAS. Moreover, SEM analyses showed that the micropyle was not occluded during oocyte storage, and ovarian fluid was observed on the surface of chilled oocytes. Therefore, Colossoma macropomum oocytes could be preserved under chilled storage at 13 °C for 30 min throughout its breeding season.