Lactococcus petauri, a lactic acid bacterium described as a new species of the Lactococcus genus, has emerged as a significant health challenge in tilapia fish farming. This bacterium has been associated with lactococcosis, necrotizing enteritis and septicemia in fish, which in many cases has been confused with L. garvieae due to their great phylogenetic similarity. Both pathogens are responsible for significant economic losses due to the high mortality of infected fish. Intensive farming of Nile tilapia requires strict monitoring of animal health in order to accurately identify pathogens. The aim of this study was to identify colonies isolated from tilapia by PCR and SANGER sequencing of the gyrB gene for L. petauri, as well as to characterize growth behaviour and antibiotic resistance (AST). The strain was seeded on tryptic soy agar (TSA) for 12 hours at 37 ºC, for antibiotic susceptibility testing using the agar diffusion method with antibiotic-impregnated disks (commercial). A bacterial suspension was made in saline solution (0.85%) to the 0.5 MacFarland scale, which was then pipetted 100 µL into Petri dishes containing TSA. The growth kinetics of the bacteria were assessed by adjusting the inoculum to OD 0.01 in tryptic soy broth (TSB) and evaluating its growth in a BioTek model Eon™ microplate reader every 15 min for 24h. The strain was confirmed by sequencing as L. petauri and showed significant growth in TSB, reaching the logarithmic phase in the first 3.5 hours and reaching the stationary phase after 6 hours of cultivation, with a final OD of 1.0 nm. In the AST test, the bacteria showed sensitivity (mm) to vancomycin 30 µg (21.4), streptomycin 10 µg (18.6), gentamicin 10 µg (19.8), azithromycin 15 µg (27.1), erythromycin 15 µg (27.5), chloramphenicol 30 µg (30.1), ampicillin 10 µg (31.5), enrofloxacin 5 µg (25.5) and ceftiofur 30 µg (35.5) with florfenicol 30 µg standing out, approved for use in aquaculture, which showed a double halo of 20.6 mm bactericidal and up to 30.2 mm bacteriostatic. It was intermediate for clindamycin 2 µg (15.9), rifampicin 5 µg (15.2), neomycin 30 µg (16.8) and sulfazotrim 25 µg was the only antibiotic with resistance. These results reinforce the importance of characterizing emerging pathogens and accurate diagnostic methods to elucidate the mechanisms of pathogenicity and develop effective approaches to mitigate their negative impacts on tilapia fish farming.