Lactococcus petauri is an emerging pathogen causing piscine lactococcosis in various aquatic species. This disease has been increasingly reported as a cause of mortality in aquaculture. Due to the indiscriminate use of antimicrobials and to promote viable alternatives for their replacement, bioactive peptides with antimicrobial properties can be explored as tools against bacterial infections. Thus, the objective of this study was to identify the pathogen responsible for a mortality outbreak in cage-cultured Nile tilapia (Oreochromis niloticus), assess its pathogenicity, and evaluate the therapeutic efficacy of a commercial bioactive peptide and florfenicol against L. petauri. The bacterium was isolated from affected Nile tilapia exhibiting external and internal hemorrhages, ocular opacity, anorexia, and ascites. Koch's postulates were fulfilled by inoculating healthy fish with 3.6 × 10