Cyperus rotundus L. has been used for pharmaceutical applications including antibacterial infections. Nevertheless, there is still no data regarding the mode of actions. This study aimed to determine the antibacterial activity and mode of actions of Cyperus rotundus extract (CRE) against ampicillin-resistant Staphylococcus aureus (ARSA) which poses a serious problem for hospitalized patients. The majority of chemical compounds of CRE were flavonoids and alkaloids. The minimum inhibitory concentrations (MICs) for ampicillin and CRE against all ARSA strains were 64 μg/ml and 0.5 mg/ml, respectively. Checkerboard assay revealed synergistic activity in the combination of ampicillin and CRE at the lowest fractional inhibitory concentration index (FICI) at 0.27. The killing curve assay had confirmed the synergistic and bactericidal activity of the combination against ARSA. Electron microscopic results showed that these ARSA cells treated with this combination caused peptidoglycan and cytoplasmic membrane (CM) damage and average cell areas significantly smaller than control. Also, this combination caused an increase in CM permeability of ARSA. CRE revealed the inhibitory activity against β-lactamase. It is normally known that some drugs are derived from flavonoids or alkaloids. So, this CRE proposes the potential to develop a novel adjunct phytopharmaceutical to ampicillin for the remedy of ARSA.