Fractions of S. rebaudiana leaves, extracted in Ethyl Acetate (EAF), rich in antioxidant compounds and Isobutanolic fraction (IF), with a high content of glycosides, were evaluated for antibacterial and antibiofilm potential against reference strains of Gram-positive and Gram-negative bacteria of clinical interest. EAF had bactericidal action against Gram-positive strains evaluated and against most Gram-negative strains, with the exception of Salmonella enteritidis. Gram-positive species showed greater sensitivity to EAF. For IF, up to the maximum concentration, there was no bactericidal effect for the strains. The pretreatment of planktonic cells of S. aureus with subinhibitory concentrations of EAF determined a reduction in the potential of structuring of the biofilm by up to 71%. The fraction showed effectiveness in biofilm treatments formed by S. aureus, with a 99% reduction in cell viability compared to the control. In addition to the antibacterial action on planktonic cells, EAF has promising effects against S. aureus biofilms.