To find more effective ways of overcoming methicillin-resistant Staphylococcus aureus (MRSA), there has been considerable interest in the use of marine-derived constituents as alternatives to control pathogenic microorganisms. In this study, we investigated whether phlorofucofuroeckol-A (PFF) isolated from the edible brown alga Eisenia bicyclis suppressed production or function of penicillin-binding protein 2a (PBP2a). The antimicrobial mode of action of PFF in MRSA was identified by measuring cell membrane integrity and using the time-kill curve method. We attempted to determine the antimicrobial effects of PFF on the expression level of the resistance determinants mecA and its regulatory genes mecI and mecR1 in MRSA by reverse transcriptase polymerase chain reaction. PFF suppressed mecI, mecR1, and mecA gene expression in a dose-dependent manner. In addition, we revealed PFF mediates the suppressive effect of PBP2a expression in MRSA by Western blot analysis. PFF suppressed production of the PBP2a protein, suggesting that PFF probably acts by controlling the methicillin resistance-associated genes involved in the cell wall and production of PBP2a. These results demonstrate that PFF isolated from E. bicyclis significantly suppressed the expression of the methicillin resistance-associated genes and production of PBP2a, which is considered the primary cause of methicillin resistance.