One of the significant obstacles to the growth of sugarcane production is the infection by phytopathogens, mainly by the bacterium Leifsonia xyli subsp. xyli (Lxx) causal agent of Ratoon stunting disease. Thus, this research aimed to evaluate the effects of kasugamycin on the in vitro growth of sugarcane, as well as its effect on the bacterium Lxx. Explants of strain SP791011 from sugarcane were inoculated in MS culture medium supplemented with the antimicrobial kasugamycin at concentrations of 0.00; 0.87; 1.08; 1.74 and 3.48 mL.L-1, where they remained for 30 days. After this period, the survival rate, shoot number per explant, height of the explants, phytomass, dry phytomass and phytosanitary were evaluated based on the presence of genomic DNA of Lxx. It was verified that the culture in kasugamycin influenced the morphological variables negatively; nevertheless, the antimicrobial did not demonstrate phytotoxicity to the plants. All treatments tested in this experiment were diagnosed as positive, with DNA amplification for Lxx, despite it was observed a reduction in bacterial load, suggesting that kasugamycin at higher doses can be evaluated as an attempt to eliminate the bacterium in the in vitro cultivation of sugarcane.