Methicillin-resistant Staphylococcus aureus (MRSA) is considered as one of the most dangerous clinical pathogens. Biofilms forming ability of MRSA is also a major cause of drug resistance. Hence, it is in urgent need to develop novel antibacterial/antibiofilm drugs. Fascaplysin with a unique cationic five-ring coplanar backbone is emerging as a potential antibacterial compound. In this study, aiming at developing novel and more effective agents, a series of fascaplysin derivatives and their corresponding β-carboline precursors have been synthesized. Then their antibacterial/antibiofilm activity and mechanisms against MRSA were investigated for the first time. The results showed that most fascaplysins rather than β-carboline precursors exhibit superior antimicrobial activity against MRSA ATCC43300, demonstrating the important role of cationic five-ring coplanar backbone playing in antibacterial activity. Among them, 14 and 18 are the most potent compounds with MIC value of 0.098 μg/ml (10-fold lower than vancomycin), and 18 featuring the lowest toxicity. Subsequent mechanisms exploration indicates that 18 has relatively stronger ability to destroy bacterial cell wall and membrane, higher binding affinity to bacterial genomic DNA. Molecular docking study revealed that besides the key role of cationic five-ring coplanar backbone, introduction of N-aryl amide at 9-position of fascaplysin promoted the combination of compound 18 and DNA via additional π-π stacking and hydrogen bonding of the naphthyl group. Moreover, fascaplysins could inhibit MRSA biofilm formation in vitro and bacterial infection in vivo. All these results illustrate that fascaplysin derivative 18 is a strong and safe multi-target antibacterial agent, which makes it an attractive candidate for the treatment of MRSA and its biofilm infections.