Two probiotics were tested as alternative treatment to limit the prevalence of the vertebral column compression syndrome (VCCS) in rainbow trout, compared with a preventive treatment with florfenicol. Either the antibiotic, or a lactic acid bacterium, Pediococcus acidilactici, or a yeast, Saccharomyces cerevisiae var. boulardii, was introduced into experimental diets, which were compared with the control diet without supplementation. The antibiotic caused some mortality during the first week after treatment, likely because of the toxicity of the compound. However, this antibiotic treatment limited the occurrence of VCCS to 3%, vs. 13% of fish affected in the control group. Pediococcus acidilactici provided the same level of protection as the antibiotic, but on condition that the treatment was applied during the 5 months of the experiment, without any adverse effect on survival. A treatment with P. acidilactici only during the first 20 days of feeding was not sufficient to limit VCCS, nor was the treatment with the yeast sufficient. These results were discussed while comparing the microbiota associated with the intestine. The experiment reinforced the hypothesis that pathogenic bacteria are involved in VCCS. The long-term dietary supplementation with P. acidilactici seemed promising as a preventive treatment against the syndrome, but large-scale investigation in fish farms will be necessary to confirm its prophylactic relevance.