This study evaluated the effects of autochthonous single‐strain and multi‐strain (mix) probiotics on the zootechnical performance and sanitary conditions of juvenile neotropical fish. Fingerlings of tambaqui (Colossoma macropomum) were fed three diets of single‐strain probiotics (two Enterococcus faecium strains and one Bacillus cereus autochthonous strain) and a multi‐strain probiotic diet (a mix of three probiotic strains) for 120 days. After dietary supplementation, 90 tambaquis were intraperitoneally injected with Aeromonas hydrophila at a concentration of 1.8 × 108 CFU·g−1. Clinical signs of disease, infectious intensity and accumulated mortality rates were evaluated. The use of diets containing probiotics, regardless of strain, enhanced productive performance from 90 experimental days (p < 0.05). The multi‐strain probiotics reduced the presence of potentially pathogenic bacteria in the intestine. Fish fed probiotics showed improved resistance to A. hydrophila infection, while the diets containing B. cereus (an autochthonous probiotic) and multi‐strain probiotic promoted the lowest mortality rates and higher leucocyte and thrombocyte counts (p < 0.05). The results revealed that the use of probiotics as a single autochthonous or multi‐strain probiotic enhanced fish growth, prevented dysbiosis and increased disease resistance.