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Objectives: Acroptilon repens (L.) DC is an Asian native plant belongs to the Asteraceae family. It has been cultivated the United States and Canada in recent years. This study investigated the effects of antibacterial, anti-biofilm, and cytotoxicity of A. repens ethanolic extract and its components. Materials and Methods: We used disk diffusion and well diffusion strategies to screen antimicrobial activity of A. repens against Staphylococcus aureus and Staphylococcus epidermidis. Then, the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of extract and fractions were determined by the small dilution broth methodology for S. aureus and S. epidermidis. The impact of extract and fractions on biofilm formation was evaluated by microtiter plate assay The MTT assay determined the toxicity effect of samples on Vero cells. The results are expressed because of the viability. Results: Results showed that A. repens extract and fragments inhibit the growth of gram-positive bacteria. The ethanolic extract and its fractions showed stronger inhibition zones for S. aureus and S. epidermidis. According to the antibiofilm test, the ethanolic extract and water fraction of A. repens have the highest antibiofilm effect on S. aureus and S. epidermidis. The value of LC50 (50% mortality) in MTT assay results was 30, 60, 30, and 2/5 mg/mL for ethanolic extract, chloroform, ethyl acetate and water fractions of A. repens, respectively. Conclusions: Extract of A. repens and its fractions have an antibacterial and antibiofilm effect on S. aureus and S. epidermidis without significant cytotoxic effect. [ABSTRACT FROM AUTHOR]